Compositions, methods, and kits for amplifying nucleic acids

ABSTRACT

The present teachings are directed to compositions, methods, and kits for amplifying target nucleic acids while reducing non-specific fluorescence and undesired amplification products, sometimes referred to as secondary amplification products or spurious side-products. The enzyme inhibitors disclosed herein comprise a nucleotide sequence and at least one quencher. Complexes comprising an enzyme inhibitor associated with an enzyme, wherein at least one enzymatic activity of the enzyme is inhibited, are also provided. Methods for amplifying a target nucleic acid while reducing undesired amplification products are disclosed, as are methods for reducing non-specific fluorescence. Kits for expediting the performance of certain disclosed methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/876,711, filed Oct. 6, 2015, which is a continuationapplication of U.S. patent application Ser. No. 13/902,742, filed May24, 2013 (now Abandoned), which is a continuation application of U.S.patent application Ser. No. 12/633,759, filed Dec. 8, 2009 (now U.S.Pat. No. 8,470,531 granted Jun. 25, 2013), which is a divisionalapplication of U.S. patent application Ser. No. 11/537,409, filed Sep.29, 2006 (now Abandoned), which claims a priority benefit under 35U.S.C. § 119(e) from U.S. Provisional Patent Application No. 60/723,383,filed Oct. 3, 2005, the contents of which are incorporated herein byreference in their entireties.

FIELD

The present teachings generally relate to compositions, methods, andkits for amplifying nucleic acids while reducing non-specificfluorescence and undesired amplification products.

INTRODUCTION

While the polymerase chain reaction (PCR) and related techniques arehighly useful for a variety of applications, the amplification ofnon-target nucleic acids due to undesired side-reactions can present asignificant problem. Such side reactions can occur as a result ofmis-priming of non-target nucleic acids and/or primer oligomerization,sometimes referred to as primer dimer formation, and the subsequentamplification of these priming artifacts. This is especially true inapplications in which PCR is carried out using a mixture of nucleicacids with significant background nucleic acids while the target nucleicacid is present in low copy number (see, e.g., Chou et al., Nucl. AcidsRes. 20:1717-1723 (1992). The generation of non-specifically amplifiedproducts has been attributed at least in part to DNA polymerase activityat ambient temperature that extends non-specifically annealed primers.(see, e.g., id.; Li et al., Proc. Natl. Acad. Sci. 87:4580 (1990).Accordingly, inhibition of DNA polymerase activity at ambienttemperature is beneficial in controlling the generation of secondaryamplicons.

Several techniques have been described which reportedly decrease theformation of undesired secondary amplification products. According tocertain “manual hot start” techniques, a component critical to DNApolymerase activity (e.g., divalent ions and/or the DNA polymeraseitself) is not added to the reaction mixture until the temperature ofthe mixture is high enough to prevent non-specific primer annealing(see, e.g., Chou et al., Nucl. Acids Res. 20:1717-1723 (1992); andD'Aquila et al., Nucl. Acids Res. 19:3749 (1991)). Less labor-intensivetechniques employ the physical separation or reversible inactivation ofat least one component of the amplification reaction. For example, themagnesium or the DNA polymerase can be sequestered in a wax bead, whichmelts as the reaction temperature increases, releasing the sequesteredcomponent only at the elevated temperature. According to othertechniques, the DNA polymerase is reversibly inactivated or modified,for example by a reversible chemical modification of the DNA polymeraseor the binding of an antibody (see, e.g., Birch et al., U.S. Pat. No.5,677,152). At elevated reaction temperatures, the chemical modificationis reversed or the antibody molecule is denatured, releasing afunctional DNA polymerase. However, some of these techniques appear tobe leaky, in that some DNA polymerase activity is detectable at lowerreaction temperatures, or they require extended exposure of the reactionmixture at high temperatures to fully activate the DNA polymerase.

Certain currently used nucleic acid amplification techniques include astep for detecting and/or quantifying amplification products thatcomprise a nucleic acid dye, for example but not limited to, SYBR® GreenI (Molecular Probes, Eugene, Oreg.), including certain real-time and/orend-point detection techniques (see, e.g., Ririe et al., Analyt.Biochem. 245:154-60 (1997). Typically the nucleic acid dye associateswith double-stranded segments of the amplification products and/orprimer-template duplexes and emit a detectable fluorescent signal at awavelength that is characteristic of the particular nucleic acid dye.Certain amplification methods comprise a detection step for evaluatingthe purity of the amplification product(s) that comprises a nucleic aciddye, for example but not limited to, post-PCR dissociation curveanalysis, also known as melting curve analysis. Since the melting curveof an amplicon is dependent on, among other things, its length andsequence, amplicons can generally be distinguished by their meltingcurves (see, e.g., Zhang et al., Hepatology 36:723-28 (2002)). Adissociation or melting curve can be obtained during certainamplification reactions by monitoring the nucleic acid dye fluorescenceas the reaction temperatures pass through the melting temperature of theamplicon(s). The dissociation of a double-stranded amplicon is observedas a sudden decrease in fluorescence at the emission wavelengthcharacteristic of the nucleic acid dye. According to certaindissociation curve analysis techniques, an amplification product isclassified as “pure” when the melting curve shows a single, consistentmelting temperature, sometimes graphically displayed as a peak on a plotof the negative derivative of fluorescent intensity versus temperature(−dF/dt vs. T). For example, the appearance of multiple peaks in such adissociation curve from a single-plex amplification typically indicatesthe presence of undesired side reaction products. When such nucleic aciddye-based amplification product detection techniques are employed, it isoften desirable to: 1) at least decrease and preferably eliminate theformation of undesired side-reaction products and 2) at least decreaseand preferably eliminate fluorescence peaks resulting from thedenaturing of double-stranded segments of other nucleic acids, i.e.,non-amplification products.

Certain other amplification techniques may also yield undesiredamplification products due to, among other things, non-specificannealing of primers, ligation probes, cleavage probes,promoter-primers, and so forth, and subsequent enzyme activity atsub-optimal temperatures. For example, while reaction components arebeing combined, often at room temperature, or while the reactioncomposition is being heated to a desired reaction temperature. At leastsome of these techniques can benefit from a reduction in backgroundfluorescence.

SUMMARY

The present teachings are directed to compositions, methods, and kitsfor amplifying target nucleic acids while reducing non-specificfluorescence and undesired amplification products, sometimes referred toin the art as secondary amplicons or spurious side-products.

Enzyme inhibitors comprising a nucleotide sequence and a quencher aredisclosed. The disclosed inhibitors are designed to inhibit at least oneenzymatic activity of an enzyme. In certain embodiments, the nucleotidesequence of the enzyme inhibitor comprises an aptamer. In someembodiments, an enzyme inhibitor comprises an aptamer that is capable offorming at least one double-stranded segment (see, e.g., Yakimovich etal., Biochem. (Mosc.) 68(2):228-35 (2003); Nickens et al., RNA 9:1029-33(2003); Nishikawa et al., Oligonucleotides 14:114-29 (2004); and Umeharaet al., J. Biochem. 137:339-74 (2005)). In some embodiments, an enzymeinhibitor comprises a multiplicity of different quenchers. In certainembodiments, the enzyme inhibitor can assume a conformation comprisingat least one double-stranded segment at a first temperature, but issingle-stranded or substantially single-stranded when heated to a secondtemperature. According to certain embodiments, an enzyme inhibitorcomprising at least one double-stranded segment can form a complex withat least one of: a DNA polymerase, including without limitation areverse transcriptase; an RNA polymerase; a cleaving enzyme, includingwithout limitation, a structure-specific nuclease; a helicase; and aligase. In certain embodiments, an enzyme inhibitor is an ineffectivesubstrate for the corresponding enzyme because the inhibitor comprises ablocking group, a nucleotide analog, an uncleavable internucleotidelinkage, or combinations thereof.

DNA polymerase inhibitors comprising a nucleotide sequence and aquencher are disclosed. Some DNA polymerase inhibitors comprise two ormore quenchers that can be the same quencher or different quenchers. Incertain embodiments, a DNA polymerase inhibitor further comprises aminor groove binder that, in some embodiments, comprises a quencher. Insome embodiments, the 3′-end of a nucleotide sequence of a DNApolymerase inhibitor is not extendible by a DNA polymerase, typicallydue to the presence of a blocking group or non-extendible nucleotide. Insome embodiments, the nucleotide sequence of a DNA polymerase inhibitorcomprises an aptamer capable of forming at least one double-strandedsegment (see, e.g., Yakimovich et al., Biochem. (Mosc.) 68(2):228-35(2003)).

Complexes comprising an enzyme and an enzyme inhibitor are provided.Certain complexes comprise: a DNA polymerase and a DNA polymeraseinhibitor; a ligase and a ligase inhibitor; an RNA polymerase and an RNApolymerase inhibitor; a cleaving enzyme and a cleaving enzyme inhibitor;or a helicase and a helicase inhibitor. Certain complexes furthercomprise a deoxyribonucleotide, a ribonucleotide, a nucleotide analog,an accessory protein, for example but not limited to a single-strandedbinding protein (SSB) or a proliferating cell nuclear antigen (PCNA), orcombinations thereof. Typically the enzyme-enzyme inhibitor complex canform at a first temperature, and while associated with the inhibitor inthe complex, at least one catalytic activity of the enzyme is inhibited.When the complex is heated to a second temperature, the complexdissociates, releasing the enzyme.

In certain embodiments, an enzyme-enzyme inhibitor complex comprises aDNA polymerase and a DNA polymerase inhibitor. In certain embodiments, aDNA polymerase-DNA polymerase inhibitor complex further comprises anucleotide triphosphate (NTP) and/or a nucleotide analog. Certaincomplex embodiments comprise a DNA polymerase inhibitor in a stem-loopconformation associated with a DNA polymerase, and optionally, a NTPand/or a nucleotide analog. Certain complex embodiments comprise a DNApolymerase associated with a DNA polymerase inhibitor comprising atleast two oligonucleotides that are annealed to form a duplex comprisingat least one double-stranded segment, and optionally, a NTP and/or anucleotide analog. Typically, the DNA synthesis activity of the DNApolymerase is inhibited when it is complexed with a DNA polymeraseinhibitor of the current teachings, and optionally, a NTP and/or anucleotide analog.

Methods for reducing non-specific fluorescence comprising the enzymeinhibitors of the present teachings are disclosed. According to certainmethods, an enzyme is contacted with an enzyme inhibitor underconditions suitable for an enzyme-enzyme inhibitor complex to form. Atleast one enzymatic activity of the enzyme is inhibited while the enzymeis in the complex. When the enzyme-enzyme inhibitor complex is heated toa suitable second temperature, the complex dissociates, releasing theenzyme.

Some methods for reducing non-specific fluorescence comprise a DNApolymerase inhibitor of the present teachings. According to certain suchmethods, a reaction composition is formed at a first temperaturecomprising: a DNA polymerase, a DNA polymerase inhibitor comprising anucleotide sequence and a quencher, a NTP and/or a nucleotide analog, atarget nucleic acid, a primer, and a nucleic acid dye. In certainembodiments, the primer comprises a primer pair. At the firsttemperature, the DNA polymerase inhibitor comprises at least onedouble-stranded segment and can form a complex with the DNA polymerase.The quencher of the DNA polymerase inhibitor can absorb at least some ofthe fluorescent signal of the nucleic acid dye associated with thedouble-stranded segment of the DNA polymerase inhibitor. The reactioncomposition is heated to a second reaction temperature that is typicallynear, at, or above the melting temperature of the DNA polymeraseinhibitor, causing at least some of the DNA polymerase inhibitor-DNApolymerase complexes to dissociate. The reaction composition issubjected to at least one cycle of amplification and a multiplicity ofamplicons is generated. The double-stranded amplicons can be detected,either in “real time” or after the amplification reaction is completed,due to the fluorescence of the nucleic acid dye associated with theamplicons, while the fluorescence of the nucleic acid dye associatedwith the double-stranded segments of the DNA polymerase inhibitors is atleast reduced by the quencher.

Methods for amplifying a target nucleic acid using the enzyme inhibitorsof the present teachings are also disclosed. According to certain suchmethods, a reaction composition is formed at a first temperaturecomprising: a DNA polymerase, a DNA polymerase inhibitor comprising anucleotide sequence and a quencher, a NTP, a target nucleic acid, aprimer, and a nucleic acid dye. In certain embodiments, the primercomprises a primer pair. At the first temperature, the DNA polymeraseinhibitor comprises at least one double-stranded segment and can form acomplex with the DNA polymerase. The quencher of the DNA polymeraseinhibitor can absorb at least some of the fluorescence emitted by thenucleic acid dye associated with the double-stranded segment of the DNApolymerase inhibitor. The reaction composition is heated to a secondreaction temperature that is typically near, at, or above the meltingtemperature of the DNA polymerase inhibitor, causing at least some ofthe DNA polymerase inhibitor-DNA polymerase complexes to dissociate. Thereaction composition is subjected to at least one cycle of amplificationand a multiplicity of amplicons is generated. In certain embodiments,the amount of amplicon that is generated is increased due to thepresence of the DNA polymerase inhibitor in the reaction composition.

According to certain methods, a reaction composition comprises a targetnucleic acid, an enzyme, an enzyme inhibitor, a nucleic acid dye, and atleast one of: a NTP, a nucleotide analog, a primer, a ligation probepair, a cleavage probe pair, a promoter-primer, a cofactor, for examplebut not limited to a substance comprising NAD+, and an accessoryprotein, including without limitation a PCNA and/or an SSB.

According to certain methods, a ligase is contacted with a ligaseinhibitor and under suitable conditions, a ligase-ligase inhibitorcomplex is formed. According to certain methods, a cleaving enzyme iscontacted with a cleaving enzyme inhibitor and under suitableconditions, a cleaving enzyme-cleaving enzyme inhibitor complex isformed. According to certain methods, a helicase is contacted with ahelicase inhibitor and under suitable conditions, a helicase-helicaseinhibitor complex is formed. According to some methods, an RNApolymerase is contacted with an RNA polymerase inhibitor and undersuitable conditions, an RNA polymerase-RNA polymerase inhibitor complexis formed.

Kits for performing certain of the instant methods are also disclosed.In some embodiments, kits comprise an enzyme inhibitor comprising anucleotide sequence and a quencher. In certain embodiments, kitscomprise two or more different enzyme inhibitors. In some embodiments,an enzyme inhibitor can form a complex with an RNA polymerase, ahelicase, a cleaving enzyme, or a ligase. Certain kit embodimentsfurther comprise a cleavage probe set, a ligation probe set, a primer, apromoter-primer, or combinations thereof.

Certain kit embodiments include at least one DNA polymerase inhibitorcomprising a nucleotide sequence and a quencher. In some embodiments, akit comprises two or more DNA polymerase inhibitors. In certainembodiments, a DNA polymerase inhibitor comprises a minor groove binder.Certain kit embodiments further comprise at least one of: a primer, aprimer pair, a nucleic acid dye, a DNA polymerase, and a reporter probe.In some embodiments, a kit comprises a DNA-dependent DNA polymerase anda reverse transcriptase.

These and other features of the present teachings are set forth herein.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. These figures are not intended tolimit the scope of the present teachings in any way.

FIGS. 1A through 1F: schematically depicts illustrative embodiments ofcertain exemplary enzyme inhibitors comprising a single oligonucleotide.

FIGS. 2A through 2E: schematically depicts illustrative embodiments ofcertain exemplary enzyme inhibitors comprising a multiplicity ofoligonucleotides.

FIG. 3: depicts dissociation curves obtained using certain exemplary DNApolymerase inhibitors, plotted as the negative derivative offluorescence (−dF/dt) versus temperature in ° C.

FIG. 4: depicts dissociation curves obtained using certain exemplary DNApolymerase inhibitors, plotted as the negative derivative offluorescence versus temperature in ° C.

FIG. 5: depicts dissociation curves obtained using certain exemplary DNApolymerase inhibitors, plotted as the negative derivative offluorescence versus temperature in ° C.

FIG. 6: depicts dissociation curves obtained using certain exemplary DNApolymerase inhibitors, plotted as the negative derivative offluorescence versus temperature in ° C.

FIG. 7: depicts a photograph of agarose gel. Aliquots of a series ofthermocycled reaction compositions comprising amplicons generated invarying concentrations of an exemplary enzyme inhibitor wereelectrophoresed in separate lanes of a non-denaturing agarose gel andvisualized with ethidium bromide, as described in Example 2. Lanes A andJ: size ladder comprising 1200 base pair, 800 base pair, 400 base pair,200 base pair, and 100 base pair size standards; lanes B-G: aliquots ofthe thermocycled reaction compositions comprising 5, 10, 25, 50, 75 or100 nM DNA polymerase inhibitor E, respectively; lane H: no templatecontrol reaction composition comprising 50 nM DNA polymerase inhibitorE; lane I: blank.

FIG. 8: depicts a photograph of an agarose gel. Aliquots of a series ofthermocycled reaction compositions comprising amplicons generated invarying concentrations of an exemplary DNA polymerase inhibitor wereelectrophoresed in separate lanes of a non-denaturing agarose gel andvisualized with ethidium bromide, as described in Example 3. Lanes A andJ: size ladder comprising 1200 base pair, 800 base pair, 400 base pair,200 base pair, and 100 base pair size standards; lanes B-H: aliquots ofthermocycled reaction compositions comprising 0, 5, 10, 25, 50, 75, or100 nM DNA polymerase inhibitor E, respectively; lane I: no templatecontrol reaction composition comprising 50 nM DNA polymerase inhibitorE.

FIG. 9: depicts a photograph of a non-denaturing agarose gel, showing adecrease in secondary amplicons due to the presence of an exemplary DNApolymerase inhibitor, as described in Example 4.

FIGS. 10A and 10B: depicts exemplary dissociation curves generatedaccording to an exemplary method of the current teachings, as describedin Example 5.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Asused in this specification, the word “a” or “an” means at least one,unless specifically stated otherwise. In this specification, the use ofthe singular includes the plural unless specifically stated otherwise.For example but not as a limitation, “a target nucleic acid” means thatmore than one target nucleic acid can be present; for example, one ormore copies of a particular target nucleic acid species, as well as twoor more different species of target nucleic acid. Also, the use of“comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are not intended tobe limiting. The term “and/or” means that the terms before and after canbe taken together or separately. For illustration purposes, but not as alimitation, “X and/or Y” can mean “X” or “Y” or “X and Y”.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature cited in this specification, including but notlimited to, patents, patent applications, articles, books, and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that any of the incorporated literaturecontradicts any term defined in this specification, this specificationcontrols. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

U.S. patent application Ser. No. 10/762,222, entitled “CompetitiveKinetic Nucleic Acid DNA polymerase Inhibitors”, by John W. Brandis,filed Jan. 11, 2004, is hereby expressly incorporated by reference inits entirety for any purpose.

Some Definitions

The term “absorb at least some of” when used in reference to thefluorescent signal emitted from a nucleic acid dye refers to thereduction of detectable fluorescence due to the presence of one or morequenchers of an enzyme inhibitor. To absorb at least some of thefluorescence emitted by the nucleic acid dye associated withdouble-stranded segments of an enzyme inhibitor means that there is ameasurable decrease in detectable fluorescence at the emissionwavelength that is characteristic of the nucleic acid dye relative tothe detectable fluorescence in a reaction composition comprising thesame components except that the enzyme inhibitor does not comprise thequencher. In some embodiments, a measurable decrease in detectablefluorescence means a 30%, a 40%, a 50%, a 60%, a 70%, an 80%, a 90%, a95%, a 97%, a 98%, a 99%, or a greater than a 99% relative decrease influorescence. In certain embodiments wherein the at least one quenchercomprises a fluorescent quencher, there can be a measurable decrease inthe detectable fluorescence at the wavelength that is characteristic ofthe nucleic acid dye and a measurable increase in the detectablefluorescence at the characteristic emission wavelength of at least onefluorescent quencher of the enzyme inhibitor.

The terms “amplicon” and “amplification product” as used hereingenerally refers to the product of an amplification reaction. Anamplicon can be double-stranded or single-stranded, and can include theseparated component strands obtained by denaturing a double-strandedamplification product. In some embodiments, an amplicon comprises aligation product (for example but not limited to a ligated probe), thecomplement of at least part of a ligation product, or both. In certainembodiments, the amplicon of one amplification cycle can serve as atemplate in a subsequent amplification cycle.

The terms “annealing” and “hybridizing”, including without limitationvariations of the root words hybridize and anneal, are usedinterchangeably and mean the nucleotide base-pairing interaction of onenucleic acid with another nucleic acid that results in the formation ofa duplex, triplex, or other higher-ordered structure. In someembodiments of the present teachings, annealing or hybridization refersto the interaction between at least some of the nucleotides in at leasttwo regions of the same enzyme inhibitor to form a hairpin or stem-loopstructure, sometimes referred to as self-annealing. The primaryinteraction is typically nucleotide base specific, e.g., A:T, A:U, andG:C, by Watson-Crick and Hoogsteen-type hydrogen bonding. In certainembodiments, base-stacking and hydrophobic interactions may alsocontribute to duplex stability. Conditions under which primers andprobes anneal to complementary sequences are well known in the art,e.g., as described in Nucleic Acid Hybridization, A Practical Approach,Hames and Higgins, eds., IRL Press, Washington, D.C. (1985) and Wetmurand Davidson, Mol. Biol. 31:349, 1968. In general, whether suchannealing takes place is influenced by, among other things, the lengthof the complementary portions of the corresponding first and thirdregions and/or fourth and sixth regions of certain enzyme inhibitors,the complementary portions of the primers and their correspondingbinding sites in the target flanking sequences and/or amplicons, thecomplementary portions of the cleavage probes or the ligation probes andthe corresponding binding portions of the target nucleic acid oramplicon, or the corresponding complementary portions or a reporterprobe and its binding site; the pH; the temperature; the presence ofmono- and divalent cations; the proportion of G and C nucleotides in thehybridizing region; the viscosity of the medium; and the presence ofdenaturants. Such variables influence the time required forhybridization. In certain enzyme inhibitor embodiments, the presence ofcertain nucleotide analogs or minor groove binders in the inhibitor,probes, and/or primers can also influence hybridization conditions.Thus, the preferred annealing conditions will depend upon the particularapplication. Such conditions, however, can be routinely determined bypersons of ordinary skill in the art, without undue experimentation.Preferably, annealing conditions are selected to allow the primersand/or probes to selectively hybridize with a complementary sequence inthe corresponding target flanking sequence or amplicon, but nothybridize to any significant degree to different target nucleic acids ornon-target sequences in the reaction composition at the second reactiontemperature.

The term “selectively hybridize” and variations thereof means that,under appropriate stringency conditions, a given sequence (for examplebut not limited to a primer) anneals with a second sequence comprising acomplementary string of nucleotides (for example but not limited to atarget flanking sequence or a primer-binding site of an amplicon), butdoes not anneal to undesired sequences, such as non-target nucleicacids, probes, or other primers. Typically, as the reaction temperatureincreases toward the melting temperature of a particular double-strandedsequence, the relative amount of selective hybridization generallyincreases and mis-priming generally decreases. In this specification, astatement that one sequence hybridizes or selectively hybridizes withanother sequence encompasses situations where the entirety of both ofthe sequences hybridize or selectively hybridize to one another, andsituations where only a portion of one or both of the sequenceshybridizes or selectively hybridizes to the entire other sequence or toa portion of the other sequence.

As used herein, the term “stringency” is used to define the temperatureand solvent composition existing during hybridization and the subsequentprocessing steps at which a hybrid comprised of two complementarynucleotide sequences will form. Stringency also defines the amount ofhomology, the conditions necessary, and the stability of hybrids formedbetween two nucleotide sequences. As the stringency conditions increase,selective hybridization is favored and non-specific cross-hybridizationis disfavored. Increased stringency conditions typically correspond tohigher incubation temperatures, lower salt concentrations, and/or higherpH, relative to lower stringency conditions at which mis-priming,including without limitation, the mis-annealing of ligation probesand/or cleavage probes, is more likely to occur. Those in the artunderstand that appropriate stringency conditions to enable theselective hybridization of a primer or primer pair, a ligation probepair, and/or a cleavage probe pair to a corresponding target flankingsequence and/or amplicon can be routinely determined using well knowntechniques and without undue experimentation (see, e.g., PCR: The Basicsfrom background to bench, McPherson and Moller, Bios ScientificPublishers (2000; hereinafter “McPherson”)).

In this specification, a statement that one nucleic acid sequence is thesame as or substantially the same as another nucleotide sequenceencompasses situations where both of the nucleotide sequences arecompletely the same as or substantially the same as the other sequence,and situations where only a portion of one of the sequences is the sameas or substantially the same as a portion of the entire other sequence.Likewise, a statement that one nucleic acid sequence is complementary toor substantially complementary to another nucleotide sequenceencompasses situations where both of the nucleotide sequences arecompletely complementary or substantially complementary to one another,and situations where only a portion of one of the sequences iscomplementary to or substantially complementary to a portion of theentire other sequence.

The term “aptamer” as used herein refers to a DNA or RNA oligonucleotidethat: 1) is typically identified originally using an in vitro selectionprocess, for example but not limited to the “systematic evolution ofligands by exponential enrichment” (SELEX) process or a variationthereof, and 2) recognizes and binds to a binding partner, for examplebut not limited to an enzyme, in a highly specific,conformation-dependent manner.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the terms “complementary” and “complementarity” are usedin reference to at least two nucleic acids that are related by thebase-pairing rules. For example but without limitation, the sequence“A-C-T” is complementary to the sequence “T-G-A.” Complementarity may bepartial, in which case only some of the nucleotides are matchedaccording to the base-pairing rules. Or, there may be complete or totalcomplementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has a significant effect on the efficiencyand strength of hybridization between the nucleic acid strands.Complementarity need not be total for a stable duplex to form, i.e.,stable duplexes may contain mismatched base pairs or unmatched bases.Those in the art can determine duplex stability empirically consideringa number of variables including without limitation, the length of thenucleic acid, base composition and sequence of the nucleic acid, ionicstrength, and incidence of mismatched base pairs. The stability of anucleic acid duplex is typically measured by its melting temperature.

As used herein, the terms “complex” and “enzyme inhibitor-enzymecomplex” refer to the association between an enzyme inhibitor of thepresent teachings and the corresponding enzyme. In some embodiments, anenzyme inhibitor-enzyme complex comprises a DNA polymerase, an RNApolymerase, a ligase, a cleaving enzyme, or a helicase. The termsinhibit, inhibits, and variations thereof, when used in reference to anenzyme, are relative terms and refer to a measurable decrease inenzymatic activity compared to the activity of the enzyme under the sameamplifying conditions but in the absence of the enzyme inhibitor. Incertain embodiments, the enzymatic activity of the enzyme is decreasedby about 40%, about 50%, about 60%, about 70%, about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, orgreater than 99%, when complexed with the enzyme inhibitor, asdetermined by the quantity of desired amplicon generated in parallelamplification reactions in the presence and the absence of the enzymeinhibitor. In certain embodiments, optimal inhibition is obtained whenthe complex further comprises an accessory protein, a NTP, a nucleotideanalog, a substance comprising NAD+, or combinations thereof.

The term “corresponding” as used herein refers to at least one specificrelationship between the elements to which the term relates. Forillustration purposes but not as a limitation, at least one forwardprimer of a particular primer pair corresponds to at least one reverseprimer of the same primer pair; at least one primer is designed toanneal with the flanking region of the corresponding target nucleic acidand/or the primer-binding portion of at least one correspondingamplicon; a first probe of a ligation probe set anneals to a targetnucleic acid and/or an amplicon upstream of, and typically adjacent to,the ligation site and the corresponding second ligation probe anneals tothe target nucleic acid and/or an amplicon downstream of, and typicallyadjacent to, the ligation site; in certain enzyme inhibitor embodiments,a first oligonucleotide anneals with the corresponding secondoligonucleotide to form a duplex comprising at least one double-strandedsegment; and so forth.

The terms “denaturing” and “denaturation” as used herein refer to anyprocess in which a double-stranded polynucleotide, including withoutlimitation, a gDNA fragment comprising at least one target nucleic acid,a double-stranded amplicon, or a polynucleotide comprising at least onedouble-stranded segment, for example but not limited to an enzymeinhibitor at a first temperature, is converted to two single-strandedpolynucleotides or to a single-stranded or substantially single-strandedpolynucleotide, as appropriate. Denaturing a double-strandedpolynucleotide or a double-stranded segment of an enzyme inhibitorincludes without limitation, a variety of thermal and chemicaltechniques which render a double-stranded nucleic acid or adouble-stranded segment of an enzyme inhibitor single-stranded orsubstantially single-stranded, for example but not limited to, releasingthe two individual single-stranded components of a double-strandedpolynucleotide or a duplex comprising two oligonucleotides. Those in theart will appreciate that the denaturing technique employed is generallynot limiting unless it substantially interferes with a subsequentannealing or enzymatic step of an amplification reaction or, in certainmethods, the detection of a fluorescent signal.

The term “double-stranded,” as used herein refers to one or two nucleicacid strands that have hybridized along at least a portion of theirlengths. Thus, in certain contexts, “double-stranded” can refer to aportion of a single oligonucleotide that can fold so that at least onesegment of the first region of the oligonucleotide hybridizes to atleast one segment of the third region of the same oligonucleotide, atleast one segment of the fourth region of the oligonucleotide hybridizeswith at least one segment of the sixth region of the oligonucleotide, orboth, thereby forming one or more double-stranded segments and one ormore single-stranded portions. Hence, a single nucleic acid strand canform hairpin or stem-loop conformations that have double-stranded andsingle-stranded segments (see, e.g., FIG. 1). Similarly, twocomplementary oligonucleotides can hybridize with each other to form aduplex (see, e.g., FIG. 2). Hence, “double-stranded” does not mean thata nucleic acid must be entirely double-stranded. Instead, adouble-stranded nucleic acid can have one or more single-strandedsegment and one or more double-stranded segment.

The term “first temperature” refers to the temperature, often a range oftemperatures, at which an enzyme-enzyme inhibitor complex can form. Theterm “second temperature” refers to the temperature, often a range oftemperatures, at which an enzyme-enzyme inhibitor complex dissociates ordoes not form. As those in the art will appreciate, the secondtemperature is typically at or near the Tm of the enzyme inhibitor,while the first temperature is typically below the Tm of the enzymeinhibitor to allow the enzyme inhibitor to assume a conformationcomprising at least one double-stranded segment. An exemplary firsttemperature can be ambient or “room temperature”.

As used herein, the term “Tm” is used in reference to meltingtemperature. The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands.

A “microfluidics device” is a reaction vessel comprising at least onemicrochannel, generally including an internal dimension of onemillimeter or less. Microfluidics devices typically employ very smallreaction volumes, often on the order of one or a few microliters (μL),nanoliters, or picoliters. Those in the art will appreciate that thesize, shape, and composition of a microfluidics device is generally nota limitation of the current teachings. Rather, any suitablemicrofluidics devices can be employed in performing one or more steps ofthe disclosed methods. Descriptions of exemplary microfluidics devicesand uses thereof can be found in, among other places, Fiorini and Chiu,BioTechniques 38:429-46 (2005); Kelly and Woolley, Analyt. Chem.77(5):96A-102A (2005); Cheuk-Wai Kan et al., Electrophoresis 25:3564-88(2004); and Yeun et al., Genome Res. 11:405-12 (2001).

The term “minor groove binder” as used herein refers to a small moleculethat fits into the minor groove of double-stranded DNA, sometimes in asequence specific manner. Generally, minor groove binders are long, flatmolecules that can adopt a crescent-like shape and thus, fit snugly intothe minor groove of a double helix, often displacing water. Minor groovebinding molecules typically comprise several aromatic rings connected bybonds with torsional freedom, for example but not limited to, furan,benzene, or pyrrole rings.

“Mis-priming” or “mis-primed,” as used herein, refer to thehybridization of a primer or a probe to a non-target nucleic acid. As isknown in the art, primers (excluding random primers) are generallydesigned to hybridize to a selected sequence that flanks a targetnucleic acid or to a primer-binding site of an amplicon and to directDNA synthesis or primer extension starting at that site. Mis-priming canoccur when a primer or a probe hybridizes to a non-target nucleic acid,oftentimes at low or decreased stringency conditions, and then serves asthe initiation point for primer extension from that non-target site,giving rise to synthesis of certain undesired secondary amplificationproducts. Ligation probe pairs and cleavage probe pairs can alsomis-anneal to a non-target nucleic acid, oftentimes at low or decreasedstringency conditions, which can also result in the formation ofundesired amplification products.

The term “non-extendable nucleotide” as used herein refers to anucleotide to which substantially no other nucleotide can be added by apolymerase. In some embodiments, the non-extendable nucleotides arenucleotide analogs that do not have optimal functional groups forformation of a phosphodiester linkage with another nucleotide. Incertain embodiments, the non-extendable nucleotides arechain-terminating nucleotides that allow essentially no primerextension, for example dideoxynucleotides (ddNs), such as ddA, ddC, ddG,ddI, ddT, and ddU. In some embodiments, a polymerase can link othernucleotides to the non-extendable nucleotide, but at slow rate.

The terms “non-specific” or “background” when used in reference tofluorescence refer to the detectable signal emitted from nucleic aciddye molecules associated with double-stranded nucleic acids other thandesired amplicons. Desired amplicons comprise the amplification productsof target nucleic acids, including in some embodiments, internalstandard or control sequences that may be included in certain reactioncompositions of the current teachings for, among other things,normalization and/or quantitation purposes. Thus, the fluorescent signalresulting from the association of nucleic acid dye molecules withspurious, secondary amplicons, often the result of mispriming,misligation, and/or primer dimer formation, is one source ofnon-specific fluorescence. Those in the art will appreciate that whenthe enzyme inhibitors of the present teachings comprise at least onedouble-stranded segment at a first temperature to which nucleic acid dyemolecules can associate, the inhibitor's quencher moiety can absorb atleast some of the detectable fluorescent signal from the associatednucleic acid dye, a second source of background, thereby reducing thenon-specific fluorescence of the reaction composition.

The term “nucleotide base”, sometimes referred to as a nitrogenous baseor a nitrogen heterocyclic base, refers to a substituted orunsubstituted aromatic ring or rings that can serve as a component of anucleotide. In certain embodiments, the aromatic ring or rings contain anitrogen atom. In certain embodiments, the nucleotide base is capable offorming Watson-Crick or Hoogsteen-type hydrogen bonds with acomplementary nucleotide base. Exemplary nucleotide bases and analogsthereof include, the naturally-occurring nucleotide bases adenine,guanine, cytosine, 5 methylcytosine, uracil, and thymine, and analogs ofthe naturally occurring nucleotide bases, including, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine,N6-Δ2-isopentenyladenine (6iA), N6-Δ2-isopentenyl-2-methylthioadenine(2ms6iA), N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see, e.g., U.S. Pat. Nos.6,143,877 and 6,127,121 and PCT Published Application WO 01/38584),ethenoadenine, indoles such as nitroindole and 4-methylindole, andpyrroles such as nitropyrrole. Non-limiting examples of nucleotide basescan be found, e.g., in Fasman, Practical Handbook of Biochemistry andMolecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla. (1989) andthe references cited therein.

The term “nucleotide” as used herein refers to a phosphate ester of anucleoside, e.g., a triphosphate ester, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. The term “nucleotide” is also used to generally refer to a setof compounds including both nucleosides and nucleotides, unlessotherwise apparent from the context. The term “nucleoside”, as usedherein, refers to a compound comprising a nucleotide base linked to theC-1′ carbon of a sugar, such as ribose, arabinose, xylose, and pyranose,and sugar analogs thereof. The sugar may be substituted orunsubstituted. Substituted ribose sugars include, but are not limitedto, those riboses in which one or more of the carbon atoms, for examplethe 2′-carbon atom, is substituted with one or more of the same ordifferent, —R, —OR, —NR₂ azide, cyanide or halogen groups, where each Ris independently H, C₁-C₆ alkyl, C₂-C₇ acyl, or C₅-C₁₄ aryl. Exemplaryriboses include, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT Published Application Nos. WO 98/22489, WO 98/39352, andWO 99/14226; and Braasch and Corey, Chem. Biol. 8:1-7, 2001; and U.S.Pat. No. 6,268,490). “LNA” or “locked nucleic acid” is a nucleotideanalog that is conformationally locked such that the ribose ring isconstrained by a methylene linkage between, for example but not limitedto, the 2′-oxygen and the 3′- or 4′-carbon or a 3′-4′ LNA with a 2′-5′backbone (see, e.g., Imanishi and Obika, U.S. Pat. No. 6,268,490; andWengel and Nielsen, U.S. Pat. No. 6,670,461). The conformationrestriction imposed by the linkage often increases binding affinity forcomplementary sequences and increases the thermal stability of suchduplexes. Exemplary LNA sugar analogs within a polynucleotide includethe structures:

where B is any nucleotide base.

The 2′- or 3′-position of ribose can be modified to include hydrogen,hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,methoxyethyl, alkoxy, phenoxy, azido, cyano, amido, imido, amino,alkylamino, fluoro, chloro and bromo. Nucleotides include the natural Doptical isomer, as well as the L optical isomer forms (see, e.g.,Garbesi et al., Nucl. Acids Res. 21:4159-65 (1993); Fujimori et al., J.Amer. Chem. Soc. 112:7436-38 (1990); Urata et al., Nucl. Acids SymposiumSer. No. 29:69-70 (1993)). When the nucleotide base is a purine, e.g., Aor G, the ribose sugar is attached to the N⁹-position of the nucleotidebase. When the nucleotide base is a pyrimidine, e.g. C, T, or U, thepentose sugar is attached to the N¹-position of the nucleotide base,except for pseudouridines, in which the pentose sugar is attached to theC5 position of the uracil nucleotide base (see, e.g., Kornberg andBaker, DNA Replication, 2^(nd) Ed. (1992), Freeman, San Francisco,Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where a is an integer from 0 to 4. In certain embodiments, a is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. The term “nucleotide 5′-triphosphate” refers to a nucleotidewith a triphosphate ester group at the 5′ position, and is sometimesdenoted as “rNTP”, or “dNTP” and “ddNTP” to particularly point out thestructural features of the ribose sugar, or generically as “NTP”. Thetriphosphate ester group may include sulfur substitutions for thevarious oxygens, e.g., α-thio-nucleotide 5′-triphosphates. Reviews ofnucleotide chemistry can be found in, among other places, Miller,Bioconjugate Chem. 1:187-91 (1990); Shabarova, Z. and Bogdanov, A.Advanced Organic Chemistry of Nucleic Acids, VCH, New York (1994); andNucleic Acids in Chemistry and Biology, 2d ed., Blackburn and Gait,eds., Oxford University Press (1996; hereinafter “Blackburn and Gait”).

The term “nucleotide analogs” refers to synthetic analogs havingmodified nucleotide base portions, modified pentose portions, and/ormodified phosphate portions, and, in the case of polynucleotides,modified internucleotide linkages, as generally described herein andelsewhere (e.g., Scheit, Nucleotide Analogs, John Wiley, New York, 1980;Englisch, Angew. Chem. Int. Ed. Engl. 30:613-29, 1991; Agarwal,Protocols for Polynucleotides and Analogs, Humana Press, 1994; and S.Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134, 1998). Generally,modified phosphate portions comprise analogs of phosphate wherein thephosphorous atom is in the +5 oxidation state and one or more of theoxygen atoms is replaced with a non-oxygen moiety, for example but notlimited to, sulfur. Some non-limiting examples of phosphate analogsinclude phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, boronophosphates, including associated counterions,e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions are present. Non-limitingexamples of modified nucleotide base portions include 5-methylcytosine(5mC); C-5-propynyl analogs, including but not limited to, C-5propynyl-C and C-5 propynyl-U; 2,6-diaminopurine, also known as 2-aminoadenine or 2-amino-dA; hypoxanthine, pseudouridine, 2-thiopyrimidine,isocytosine (isoC), 5-methyl isoC, and isoguanine (isoG; see, e.g., U.S.Pat. No. 5,432,272). Non-limiting examples of modified pentose portionsinclude LNA analogs including without limitation Bz-A-LNA,5-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA (see, e.g., The Glen Report, 16(2):5(2003); Koshkin et al., Tetrahedron 54:3607-30 (1998)), and 2′- or3′-modifications where the 2′- or 3′-position is hydrogen, hydroxy,alkoxy (e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxyand phenoxy), azido, amino, alkylamino, fluoro, chloro, or bromo.Modified internucleotide linkages include phosphate analogs, analogshaving achiral and uncharged intersubunit linkages (e.g., Sterchak, E.P. et al., Organic Chem. 52:4202 (1987)), and uncharged morpholino-basedpolymers having achiral intersubunit linkages (see, e.g., U.S. Pat. No.5,034,506). Some non-limiting examples of internucleotide linkageanalogs include morpholidate, acetal, and polyamide-linked heterocycles.In one class of nucleotide analogs, known as peptide nucleic acids,including without limitation pseudocomplementary peptide nucleic acids(collectively “PNA”), a conventional sugar and internucleotide linkagehas been replaced with a 2-aminoethylglycine amide backbone polymer(see, e.g., Nielsen et al., Science, 254:1497-1500 (1991); Egholm etal., J. Am. Chem. Soc., 114: 1895-1897 (1992); Demidov et al., Proc.Natl. Acad. Sci. 99:5953-58 (2002); Peptide Nucleic Acids: Protocols andApplications, Nielsen, ed., Horizon Bioscience (2004)). A wide range ofnucleotide analogs for use in enzymatic incorporation or chemicalsynthesis are available as triphosphates, phosphoramidates, or CPGderivatives from, among other sources, Glen Research, Sterling, Md.;Link Technologies, Lanarkshire, Scotland, UK; and TriLinkBioTechnologies, San Diego, Calif. Descriptions of oligonucleotidesynthesis and certain nucleotide analogs, can be found in, among otherplaces, S. Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134 (1999);Goodchild, Bioconj. Chem. 1:165-87 (1990); Current Protocols in NucleicAcid Chemistry, Beaucage et al., eds., John Wiley & Sons, New York,N.Y., including updates through August 2005 (hereinafter “Beaucage etal.”); and Blackburn and Gait.

As used herein, the term “primer-binding site” refers to a region of apolynucleotide sequence, typically a target nucleic acid and/or anamplicon that can serve directly, or by virtue of its complement, as thetemplate upon which a primer can anneal for any suitable primerextension reaction known in the art, for example but not limited to,PCR. It will be appreciated by those of skill in the art that when twoprimer-binding sites are present on a single polynucleotide, theorientation of the two primer-binding sites is generally different. Forexample, one primer of a primer pair is complementary to and canhybridize with to the first primer-binding site, while the correspondingprimer of the primer pair is designed to hybridize with the complementof the second primer-binding site. Stated another way, in someembodiments the first primer-binding site can be in a sense orientation,and the second primer-binding site can be in an antisense orientation. Aprimer-binding site of an amplicon may, but need not comprise the samesequence as or at least some of the sequence of the target flankingsequence or its complement.

Those in the art understand that as a target nucleic acid and/or anamplification product is amplified by certain amplification means, thecomplement of the primer-binding site is synthesized in thecomplementary amplicon or the complementary strand of the amplicon.Thus, it is to be understood that the complement of a primer-bindingsite is expressly included within the intended meaning of the termprimer-binding site, as used herein.

As used herein, the term “probe-binding site” refers to a region of apolynucleotide sequence, typically a target nucleic acid and/or anamplicon that can serve directly, or by virtue of its complement, as thetemplate upon which probe can anneal. It will be appreciated by those ofskill in the art that the probe-binding site for a ligation probe paircomprise an upstream probe-binding site and a downstream probe bindingsite and that these two sites are typically adjacent to each other. Incertain embodiments, the upstream ligation probe-binding site and thedownstream probe-binding site are not adjacent to each other and anamplifying step can comprises a gap-filling reaction. It will also beappreciated by those of skill in the art that the probe-binding site fora cleavage probe pair comprises an upstream probe-binding site that isadjacent to, and may but need not overlap at least part of thedownstream cleavage probe-binding site.

Those in the art understand that as a target nucleic acid and/or anamplification product is amplified by certain amplification means, thecomplement of the probe-binding site is synthesized in the complementaryamplicon or the complementary strand of the amplicon. Thus, it is to beunderstood that the complement of a probe-binding site is expresslyincluded within the intended meaning of the term probe-binding site, asused herein.

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably and refer to single-stranded anddouble-stranded polymers of nucleotide monomers, including withoutlimitation 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA)linked by internucleotide phosphodiester bond linkages, orinternucleotide analogs, and associated counter ions, e.g., H⁺, NH₄ ⁺,trialkylammonium, Mg²⁺, Na⁺, and the like. A polynucleotide may becomposed entirely of deoxyribonucleotides, entirely of ribonucleotides,or chimeric mixtures thereof and can include nucleotide analogs. Thenucleotide monomer units may comprise any of the nucleotides describedherein, including, but not limited to, nucleotides and/or nucleotideanalogs. Polynucleotides typically range in size from a few monomericunits, e.g. 5-40 when they are sometimes referred to in the art asoligonucleotides, to several thousands of monomeric nucleotide units.Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U” denotes deoxyuridine, unless otherwise noted.

The term “quencher” as used herein refers to a moiety that absorbs atleast some of the intensity of a fluorescent emission. Quenchers can becategorized as fluorescent quenchers and dark quenchers (sometimes alsoreferred to as non-fluorescent quenchers). A fluorescent quencher is amoiety, typically a fluorophore, that can absorb the fluorescent signalemitted from a source of fluorescence at a first wavelength, for examplebut not limited to, a nucleic acid dye associated with a double-strandedsegment of nucleic acid, and after absorbing enough fluorescent energy,the fluorescent quencher can emit fluorescence at a second wavelengththat is characteristic of the quencher, a process termed “fluorescentresonance energy transfer” or FRET. For example but not as a limitation,the FAM fluorophore associated with a TAMRA fluorescent quencher can beilluminated at 492 nm, the excitation peak for FAM, and emitfluorescence at 580 nm, the emission peak for TAMRA. A dark quencher,appropriately paired with a source of fluorescence, absorbs thefluorescent energy from the source, but does not itself fluoresce.Rather, the dark quencher dissipates the absorbed energy, typically asheat. In certain embodiments, a dark quencher comprises a chromophorethat acts as an energy transfer acceptor from a fluorescent source, suchas a nucleic acid dye associated with a double-stranded segment of anenzyme inhibitor of the present teachings, but does not emit adetectable fluorescent signal of its own. Non-limiting examples of darkor non-fluorescent quenchers include DABCYL(4-(4′-dimethylaminophenylazo) sulfonic acid); Black Hole Quenchersseries quenchers, for example but not limited to BHQ-1, BHQ-2, andBHQ-3; Iowa Black; QSY series quenchers, for example but not limited toQSY-7; AbsoluteQuencher; Eclipse non-fluorescent quencher; nanocrystalsfor example but not limited to quantum dots; metals such as goldnanoparticles; and the like.

As used herein, the term “reaction vessel” generally refers to anycontainer, chamber, device, or assembly, in which a reaction can occurin accordance with the present teachings. In some embodiments, areaction vessel can be a microtube, for example but not limited to a 0.2mL or a 0.5 mL reaction tube such as a MicroAmp® Optical tube (AppliedBiosystems) or a micro-centrifuge tube, or other containers of the sortin common practice in molecular biology laboratories. In someembodiments, a reaction vessel comprises a well of a multi-well plate, aspot on a glass slide, or a channel or chamber of a microfluidicsdevice, including without limitation an Applied Biosystems TaqMan LowDensity Array. For example but not as a limitation, a plurality ofreaction vessels can reside on the same support. In some embodiments,lab-on-a-chip like devices, available for example from Caliper andFluidgm, can serve as reaction vessels in the disclosed methods. It willbe recognized that a variety of reaction vessels are commerciallyavailable or can be designed for use in the context of the presentteachings.

The term “reporter group” is used in a broad sense herein and refers toany identifiable tag, label, or moiety.

The term “small RNA molecule” is used in a broad sense herein and refersto any nucleic acid sequence comprising ribonucleotides that arenon-coding and typically have a length of: 150 nucleotides or less, 100nucleotides or less, 75 nucleotides or less, 30 nucleotides or less,between 19 and 27 nucleotides, and between 21 and 23 nucleotides. Asmall RNA molecule can be single-stranded, double-stranded, or cancomprise at least one single-stranded region and at least onedouble-stranded region, including without limitation, stem-loop orhairpin structures. Non-limiting examples of small RNA molecules includeuntranslated functional RNA, non-coding RNA (ncRNA), small non-messengerRNA (snmRNA), small interfering RNA (siRNA), tRNA, tiny non-coding RNA(tncRNA), small modulatory RNA (smRNA), snoRNA, stRNA, snRNA, microRNA(miRNA) including without limitation miRNA precursors such as primarymiRNA (pri-miRNA) and precursor miRNA (pre-miRNA), and small interferingRNA (siRNA) (see, e.g., Eddy, Nature Reviews Genetics 2:919-29 (2001);Storz, Science 296:1260-63 (2002); Buckingham, Horizon Symposia:Understanding the RNAissance:1-3 (2003)). In certain embodiments, atarget nucleic acid comprises a small RNA molecule. Those enzymeinhibitors of the current teachings that comprise ribonucleotides and/orribonucleotide analogs are expressly excluded from the intended scope ofthe term small RNA molecule as used in this specification.

The term “thermostable” when used in reference to an enzyme, indicatesthat the enzyme is functional or active (i.e., can perform catalysis) atan elevated temperature, for example but not limited to, at about 55° C.or higher. Thermostable enzymes that may be suitable for use in thecurrent teachings are commercially available from various vendors,including without limitation, Applied Biosystems (Foster City, Calif.),Promega (Madison, Wis.), Stratagene (LaJolla, Calif.), and New EnglandBioLabs (Beverly, Mass.). Those in the art will understand thatthermostable enzymes can be isolated from a variety of thermophilicand/or hyperthermophilic organisms, for example but not limited to,certain species of eubacteria and archaea, including without limitation,certain viruses that infect such organisms and that such thermostableenzymes may be suitable for use in the disclosed complexes, methods, andkits.

The terms “universal base” or “universal nucleotide” are generally usedinterchangeably herein and refer to a nucleotide analog that cansubstitute for more than one species of naturally-occurring nucleotidein a polynucleotide, including without limitation, an enzyme inhibitor.Universal bases typically contain an aromatic ring moiety that may ormay not contain nitrogen atoms and generally use aromatic ring stackingto stabilize a duplex. In certain embodiments, a universal base may becovalently attached to the C-1′ carbon of a pentose sugar to make auniversal nucleotide. In certain embodiments, a universal base does nothydrogen bond specifically with another nucleotide base. In certainembodiments, a nucleotide base may interact with adjacent nucleotidebases on the same nucleic acid strand by hydrophobic stacking.Non-limiting examples of universal nucleotides and universal basesinclude deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyriltriphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate(dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxyImPytriphosphate (dImPyTP), deoxyPP triphosphate (dPPTP),deoxypropynyl-7-azaindole triphosphate (dP7AITP), 3-methylisocarbostyril (MICS), 5-methyl isocarbyl (5MICS),imidazole-4-carboxamide, 3-nitropyrrole, 5-nitroindole, hypoxanthine,inosine, deoxyinosine, 5-fluorodeoxyuridine, 4-nitrobenzimidazole, andcertain PNA-bases, including without limitation certainpseudocomplementary PNA (pcPNA) bases. Descriptions of universal basescan be found in, among other places, Loakes, Nucl. Acids Res. 29:2437-47(2001); Berger et al., Nucl. Acids Res. 28:2911-14 (2000); Loakes etal., J. Mol. Biol. 270:426-35 (1997); Verma and Eckstein, Ann. Rev.Biochem. 67:99-134 (1998); Published PCT Application No. US02/33619, andPatron and Pervin, U.S. Pat. No. 6,433,134.

When two different oligonucleotides anneal to different regions of thesame linear complementary nucleic acid, and the 3′-end of oneoligonucleotide faces or opposes the 5′-end of the otheroligonucleotide, the former may be referred to as the “upstream”oligonucleotide and the latter the “downstream” oligonucleotide.

Certain Exemplary Components

The term “cleaving enzyme” refers to any polypeptide that can, whencombined with a nucleic acid cleavage structure (sometimes referred toas an overlap flap structure or an invasive cleavage reaction substrate)and under appropriate conditions, cleave the non-annealed flap portionof the downstream cleavage probe to generate a structure comprising aligatable nick. Non-limiting examples of cleaving enzymes includestructure-specific nucleases, for example but not limited to, certainDNA polymerases from bacteria and bacteriophages, including isolated5′exonuclease domains thereof; Cleavase® enzymes (Third WaveTechnologies, Inc., Madison, Wis.); eukaryotic flap endonucleases; andarchaeal flap endonucleases (see, e.g., Lyamichev et al., Science260:778-83 (1993); Li et al., J. Biol. Chem. 270:22109-12 (1995); Wu etal., Nucl. Acids Res. 24:2036-43 (1996); Hosfield et al., J. Biol. Chem.273:27154-61 (1998); Kaiser et al., J. Biol. Chem. 274:21387-94 (1999);Allawi et al., J. Mol. Biol. 328:537-54 (2003); and U.S. Pat. Nos.5,614,402 and 6,706,471).

A nucleic acid cleavage structure typically comprises a template strand(generally a target nucleic acid, a single-stranded amplicon, or aseparated strand of a double-stranded amplicon) hybridized with acleavage probe pair comprising two overlapping probes that hybridizewith the template strand to form a “flap”. The first or upstreamcleavage probe comprises a sequence that is complementary with a firstportion of the template strand and overlaps the 5′-end of thetemplate-complementary sequence of the second or downstream cleavageprobe, which comprises (1) a sequence that is complementary with asecond portion of the template strand that is adjacent to the firstportion of the template strand and (2) a 5′-region comprising at leastone nucleotide that may or may not be complementary with the templatestrand, but when hybridized with the template strand, is displaced bythe 3′-end of the upstream cleavage probe (see, e.g., Lyamichev et al.,Nat. Biotechnol. 17:292-96 (1999), particularly FIG. 1; Neville et al.,BioTechniques 32:S34-43 (2002), particularly FIG. 2A; Allawi et al., J.Mol. Biol. 328:537-54 (2003), particularly FIG. 2; and Brow et al., U.S.Pat. No. 6,706,471, for example at FIGS. 32 and 65). Certain cleavingenzyme inhibitors of the present teachings are designed to assume aconformation at a first temperature that resembles or mimics a nucleicacid cleavage structure. Certain disclosed cleaving enzyme inhibitorscan form a nucleic acid cleavage structure at a first temperature, butat least one oligonucleotide comprises at least one nucleotide analogand/or at least one internucleotide linkage that can not be cleaved oris slowly cleaved by the cleaving enzyme (an “uncleavableinternucleotide linkage”). Non-limiting examples of uncleavableinternucleotide linkages include phosphorothioates, including withoutlimitation phosphorodithioates; methyl phosphonates; phosphoramidates;and boranophosphates.

A “ligase” is a polypeptide that, under appropriate conditions,catalyzes phosphodiester bond formation between the 3′-OH and the5′-phosphate of adjacently hybridized probes, including withoutlimitation, a first and second ligation probe of a ligation probe set ora first cleavage probe and the hybridized fragment of a second cleavageprobe that has been cleaved by a cleaving enzyme. Temperature sensitiveligases, include but are not limited to, bacteriophage T4 ligase and E.coli ligase. Non-limiting examples of thermostable ligases include Afuligase, Taq ligase, Tfl ligase, Mth ligase, Tth ligase, Tth HB8 ligase,Tsc ligase, Thermus species AK16D ligase, Ape ligase, Lig_(Tk) ligase,Aae ligase, Rm ligase, and Pfu ligase (see, e.g., Housby et al., Nucl.Acids Res. 28:e10, 2000; Tong et al., Nucl. Acids Res. 28:1447-54, 2000;Nakatani et al., Eur. J. Biochem. 269:650-56, 2002; and Sriskanda etal., Nucl. Acids Res. 11:2221-28, 2000). The skilled artisan willappreciate that any number of mesophilic, thermostable, and/orhyperthermophilic ligases, including DNA ligases and RNA ligases, can beobtained from mesophilic, thermophilic, or hyperthermophilic organisms,for example, certain species of eubacteria and archaea, and includingcertain viruses that infect such mesophilic, thermophilic, orhyperthermophilic organisms; and that such ligases may be suitable inthe disclosed complexes, methods and kits.

The term “nucleic acid dye” as used herein refers to a fluorescentmolecule that is specific for a double-stranded polynucleotide or thatat least shows a substantially greater fluorescent enhancement whenassociated with double-stranded polynucleotide acid than with asingle-stranded polynucleotide. Typically nucleic acid dye moleculesassociate with double-stranded segments of polynucleotides byintercalating between the base pairs of the double-stranded segment, bybinding in the major or minor grooves of the double-stranded segment, orboth. Non-limiting examples of nucleic acid dyes include ethidiumbromide, DAPI, Hoechst derivatives including without limitation Hoechst33258 and Hoechst 33342, intercalators comprising a lanthanide chelate(for example but not limited to a nalthalene diimide derivative carryingtwo fluorescent tetradentate diketone-Eu3+ chelates (NDI-(BHHCT-Eu³⁺)₂),see, e.g., Nojima et al., Nucl. Acids Res. Supplemnent No. 1, 105-06(2001)), ethidium bromide, and certain unsymmetrical cyanine dyes suchas SYBR Green®, PicoGreen®, and BOXTO.

The nucleic acid sequences of certain disclosed enzyme inhibitorscomprise an aptamer. Aptamers bind target molecules in a highlyspecific, conformation-dependent manner, typically with very highaffinity, although those in the art will understand that aptamers withlower binding affinity can be selected if desired. Aptamers have beenshown to distinguish between targets based on very small structuraldifferences such as the presence or absence of a methyl or hydroxylgroup and certain aptamers can distinguish between D- and L-enantiomers.Aptamers have been obtained that bind small molecular targets, includingdrugs, metal ions, and organic dyes, peptides, biotin, and proteins,including but not limited to streptavidin, VEGF, viral proteins, andvarious enzymes, including without limitation DNA-dependent DNApolymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase,helicase, and protease (see, e.g., Lin and Jayasena, J. Mol. Biol.271:100-11 (1997); Thomas et al., J. Biol. Chem. 272:27980-86 (1997);Kulbachinskiy et al., Eur. J. Biochem. 271:4921-31 (2004); Hannoush etal., Chembiochem. 5:527-33 (2004); Bellecave et al., Oligonucleotides13:455-63 (2003); and Nishikawa et al., Nucl. Acids Res. 31:1935-43(2003)). Aptamers have been shown to retain functional activity afterbiotinylation, fluorescein labeling, and when attached to glass surfacesand microspheres.

Aptamers, including speigelmers, are identified by an in vitro selectionprocess, for example but not limited to the process known as systematicevolution of ligands by exponential amplification (SELEX). In the SELEXprocess very large combinatorial libraries of oligonucleotides, forexample 10¹⁴ to 10¹⁵ individual sequences, often as large as 60-100nucleotides long, are routinely screened by an iterative process of invitro selection and amplification. Most targets are affinity enrichedwithin 8-15 cycles and the process has been automated allowing forfaster aptamer isolation. The skilled artisan will understand thataptamers can be obtained following conventional procedures and withoutundue experimentation. Descriptions of aptamers and their selection canbe found in, among other places, L. Gold, J. Biol. Chem.,270(23):13581-84 (1995); L. Gold et al., Ann. Rev. Biochem. 64:763-97(1995); Wilson and Szostak, Ann. Rev. Biochem. 68:611-47 (1999); Cox etal., Nucl. Acids Res. 30:e108 (2002); Hermann and Patel, Science287:820-25 (2000); Vuyisich and Beal, Chem. & Biol. 9:907-13 (2002); S.Jayasena, Clin. Chem., 45:1628-50 (1999); Cox and Ellington, Bioorg.Med. Chem. 9:2525-31 (2001); Eulberg et al., Nucl. Acids Res. 33:e5(2005); and Jayasena and Gold, U.S. Pat. No. 6,183,967.

The term “DNA polymerase” is used in a broad sense herein and refers toany polypeptide that can catalyze the 5′-3′extension of a hybridizedprimer by the addition of deoxyribonucleotides and/or certain nucleotideanalogs in a template-dependent manner. For example but not limited to,the sequential addition of deoxyribonucleotides to the 3′-end of aprimer that is annealed to a nucleic acid template during a primerextension reaction. Non-limiting examples of DNA polymerases includeRNA-dependent DNA polymerases, including without limitation reversetranscriptases, and DNA-dependent DNA polymerases. It is to beappreciated that certain DNA polymerases (for example but not limited tocertain eubacterial Type A DNA polymerases and Taq DNA polymerase) mayfurther comprise a structure-specific nuclease activity and that when anamplification reaction comprises an invasive cleavage reaction, forexample but not limited to, FEN-LCR or PCR-FEN (see, e.g., Bi et al.,U.S. Pat. No. 6,511,810; and Neville et al., BioTechniques 32:S34-43(2002)), wherein the cleaving enzyme comprises a DNA polymerase, suchpolymerase is referred to herein as a cleaving enzyme in the invasivecleavage context and the corresponding enzymatic activity comprisesstructure-specific oligonucleotide cleavage. In certain embodiments, aDNA polymerase provides both a polymerization activity and astructure-specific cleaving activity. The term “RNA polymerase” refersto a DNA-dependent RNA polymerase or an RNA-dependent polymerase(sometimes referred to as an RNA replicase), and includes anypolypeptide that can catalyze the 5′-3′ addition of ribonucleotides in atemplate-dependent manner. In certain embodiments, an RNA polymerasebinds to a promoter sequence and catalyzes transcription. Non-limitingexamples of RNA polymerases include the RNA polymerases from thebacteriophages T3, T7, SP6, f2, MS2, and Qβ.

The term “primer” refers to a polynucleotide, generally anoligonucleotide comprising a “target” binding portion that is typicallyabout 12 to about 35 nucleotides long, that is designed to selectivelyhybridize with a target nucleic acid flanking sequence or to acorresponding primer-binding site of an amplification product underappropriate stringency conditions; and serve as the initiation point forthe synthesis of a nucleotide sequence that is complementary to thecorresponding polynucleotide template from its 3′-end.

The terms “forward” and “reverse” when used in reference to the primersof a primer pair indicate the relative orientation of the primers on apolynucleotide sequence. For illustration purposes but not as alimitation, consider a single-stranded polynucleotide drawn in ahorizontal, left to right orientation with its 5′-end on the left. The“reverse” primer is designed to anneal with the downstreamprimer-binding site at or near the “3′-end” of this illustrativepolynucleotide in a 5′ to 3′ orientation, right to left. Thecorresponding “forward primer is designed to anneal with the complementof the upstream primer-binding site at or near the “5′-end” of thepolynucleotide in a 5′ to 3′ “forward” orientation, left to right. Thus,the reverse primer comprises a sequence that is complementary to thereverse or downstream primer-binding site of the polynucleotide and theforward primer comprises a sequence that is the same as or substantiallythe same as the forward or upstream primer-binding site. It is to beunderstood that the terms “3-end” and “5′-end” as used in this paragraphare illustrative only and do not necessarily refer literally to therespective ends of the polynucleotide. Rather, the only limitation isthat the reverse primer of this exemplary primer pair anneals with areverse primer-binding site that is downstream of the forwardprimer-binding site that comprises the same sequence or substantiallythe same sequence as the “target” binding portion of the correspondingforward primer. As will be recognized by those of skill in the art,these terms are not intended to be limiting, but rather to provideillustrative orientation in a given embodiment.

A “primer pair” of the current teachings comprises a forward primer anda corresponding reverse primer. The forward primer comprises a firsttarget-specific portion that comprises a sequence that is the same as orsubstantially the same as the nucleotide sequence of the first orupstream target flanking sequence, and that is designed to selectivelyhybridize with the complement of the upstream target flanking sequencethat is present in, among other places, the reverse amplificationproduct. The reverse primer of the primer pair comprises a secondtarget-specific portion that comprises a sequence that is complementaryto or substantially complementary to, and that is designed toselectively hybridize with, the second or downstream target regionflanking sequence that is present in among other places, the forwardamplification product. In certain embodiments, a forward primer, areverse primer, or a forward primer and a reverse primer of a primerpair further comprises a reporter-probe binding site, a universalprimer-binding site, and/or a reporter group, for example but notlimited to a fluorescent reporter group. In some embodiments, asequencing primer comprises a fluorescent reporter group. In certainembodiments, a forward primer and the corresponding reverse primer of aprimer pair have different melting temperatures to permittemperature-based asymmetric PCR.

A universal primer or primer set may be employed according to certainembodiments of the current teachings. In certain embodiments, auniversal primer or a universal primer set hybridizes with and can beused to amplify two or more different target nucleic acid species and/ortwo or more different species of desired amplicon.

The term “probe” refers to a polynucleotide that comprises a portionthat is designed to hybridize in a sequence-specific manner with acomplementary probe-binding site on a particular nucleic acid sequence,for example but not limited to a target nucleic acid or an amplificationproduct. In certain embodiments, corresponding probes of a ligationprobe set are ligated together to form a ligated probe. In someembodiments, corresponding probes of a cleavage probe set anneal with atemplate strand to form a nucleic acid cleavage structure, which can becleaved by an appropriate cleaving enzyme under suitable conditions toform a hybridization structure comprising the template strand, theupstream cleavage probe, and a hybridized fragment of the secondcleavage probe. In certain embodiments, the annealed upstream cleavageprobe and the hybridized fragment of the downstream cleavage probe areligated together to form a ligated probe. In certain embodiments, aprobe comprises a reporter group, for example but not limited to, areporter probe. In some embodiments, a probe comprises a primer-bindingsite.

The sequence-specific portions of probes and primers of the currentteachings are of sufficient length to permit specific annealing tocomplementary sequences in target nucleic acids and desired amplicons.Detailed descriptions of primer and probe design can be found in, amongother places, Dieffenbach and Dveksler, PCR Primer, A Laboratory Manual,Cold Spring Harbor Press (1995; hereinafter “PCR Primer”); R. Rapley,The Nucleic Acid Protocols Handbook (2000), Humana Press, Totowa, N.J.(hereinafter “Rapley”); Schena; and Kwok et al., Nucl. Acid Res.18:999-1005 (1990). Primer and probe design software programs are alsocommercially available, including without limitation, Primer Express,Applied Biosystems, Foster City, Calif.; Primer Premier and BeaconDesigner software, PREMIER Biosoft International, Palo Alto, Calif.;Primer Designer 4, Sci-Ed Software, Durham, N.C.; Primer Detective,ClonTech, Palo Alto, Calif.; Lasergene, DNASTAR, Inc., Madison, Wis.;Oligo software, National Biosciences, Inc., Plymouth, Minn.; iOligo,Caesar Software, Portsmouth, N.H.; and RTPrimerDB on the world wide webat realtimeprimerdatabase.ht.st or atmedgen31.urgent.be/primerdatabase/index (see also, Pattyn et al., Nucl.Acid Res. 31:122-23 (2003)).

The skilled artisan will appreciate that the complement of the disclosedprobes and primers, target nucleic acids, desired amplicons, orcombinations thereof, may be employed in certain embodiments of thecurrent teachings. For example, without limitation, a genomic DNA samplemay comprise both the target nucleic acid sequence and its complement.Thus, in certain embodiments, when a genomic sample is denatured, boththe target nucleic acid and its complement are present in the sample assingle-stranded sequences. In certain embodiments, a primer, a ligationprobe pair, a cleavage probe pair, or combinations thereof may bedesigned to selectively hybridize to an appropriate sequence, includingwithout limitation, a target nucleic acid, the complement of a targetnucleic acid, an amplicon, and/or the complement of an amplicon.

The term “reporter probe” refers to a sequence of nucleotides and/ornucleotide analogs, that anneals with a target nucleic acid and/or anamplicon, and when detected, including but not limited to a change inintensity or of emitted wavelength, is used to identify and/or quantifythe corresponding target nucleic acid in an end-point or real-timedetection technique, for example but not limited to a Q-PCR technique.Most reporter probes can be categorized based on their mode of action,for example but not limited to: nuclease probes, including withoutlimitation TaqMan® probes (see, e.g., Livak, Genetic Analysis:Biomolecular Engineering 14:143-149 (1999); Yeung et al., BioTechniques36:266-75 (2004)); extension probes such as scorpion primers, Lux™primers, Amplifluors, and the like; hybridization probes such asmolecular beacons, Eclipse probes, light-up probes, pairs ofsingly-labeled reporter probes, hybridization probe pairs, and the like;or combinations thereof. In certain embodiments, reporter probescomprise a PNA, an LNA, a universal base, or combinations thereof, andcan include stem-loop and stem-less reporter probe configurations.Certain reporter probes are singly-labeled, while other reporter probesare doubly-labeled. Dual probe systems that comprise FRET betweenadjacently hybridized probes are within the intended scope of the termreporter probe (see, e.g., Zhang et al., Hepatology 36:723-28 (2003)).

An “unsymmetrical cyanine dye”, sometimes described in the art as anasymmetric cyanine dye or an asymmetrical cyanine dye, refers to a dyemolecule with the general formula R₂N[CH═CH]_(n)CH═NR₂, where n is asmall number and the R groups typically comprise at least one benzazolegroup and at least one quinoline group or at least one pyridine group.Non-limiting examples of unsymmetrical cyanine dyes include[2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium](SYBR® Green),[2-[N-bis-(3-dimethylaminopropyl)-amino)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium](PicoGreen®),4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridiniumiodide (BEBO), BOXTO, and BETO. Descriptions of unsymmetrical cyaninedyes can be found in, among other places, Karlsson et al., Nucl. AcidsRes. 31:6227-34 (2003); Zipper et al., Nucl. Acids Res. 32:e103 (2004);Bengtsson et al., Nucl. Acids Res. 31:e45 (2003); and Goransson et al.,Asymmetric cyanine dyes, DNA-Technology 2005, Chalmers UniversityTechnology (2005; available on the world wide web at:molbiotech.Chalmers.se/research/mk/Asymmetric%cyanine%dyes.doc).

The term “target nucleic acid” or “target” refers to the nucleic acidsequence that is specifically amplified and/or detected using thecompositions, methods, and kits of the present teachings (in contrast toa secondary amplification product, which is the result of a spuriousside-reaction, typically due to mis-priming). In certain embodiments, atarget nucleic acid serves as a template in a primer extension reaction.In some embodiments, a target nucleic acid serves as a ligationtemplate. In some embodiments, a target nucleic acid serves as atemplate strand in a nucleic acid cleavage structure. In certainembodiments, the target nucleic acid comprises DNA and is present ingenomic DNA (gDNA) or mitochondrial DNA (mtDNA). In certain embodiments,the target nucleic acid comprises RNA, for example but not limited to,ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), or anRNA molecule such as a miRNA precursor, including without limitation, apri-miRNA, a pre-miRNA, or a pri-miRNA and a pre-miRNA. In someembodiments, the target nucleic acid comprises a small RNA molecule,including without limitation, a miRNA, a siRNA, a stRNA, a snoRNA, orother ncRNA. The target nucleic acid need not constitute the entirety ofa nucleic acid molecule. For example but not as a limitation, a largenucleic acid, for example a gDNA fragment, can comprise a multiplicityof different target nucleic acids. Typically, a target nucleic acid hasat least one defined end. In many nucleic acid amplification reactionsthe target has two defined ends.

In certain embodiments, a target nucleic acid is located between twoflanking sequences, a first target flanking sequence and a second targetflanking sequence, located on either side of, but not necessarilyimmediately adjacent to, the target nucleic acid. In some embodiments, apolynucleotide such as a gDNA fragment comprises a plurality ofdifferent target nucleic acids. In some embodiments, a target nucleicacid is contiguous with or adjacent to one or more different targetnucleic acids. In some embodiments, a given target nucleic acid canoverlap one target nucleic acid on its 5′-end, another target nucleicacid on its 3′-end, or both. In other embodiments, for example but notlimited to when the target comprises a small RNA molecule, the targetmay not comprise a flanking region and a primer is designed to annealwith a portion of the small RNA target, typically an end of the targetnucleic acid (see, e.g., Chen et al., U.S. patent application Ser. No.10/947,460.

Certain Exemplary Component Techniques

According to the instant teachings, a target nucleic acid may beobtained from any living or once living organism, including aprokaryote, an archaea, or a eukaryote, for example but not limited to:an insect, including without limitation Drosophila; a worm, includingwithout limitation C. elegans; a plant, including without limitationArabidopsis; and an animal, including without limitation a human, amouse, a domesticated animal, or a non-human primate; and includingprokaryotic cells and cells, tissues, and organs obtained from aeukaryote, for example but not limited to, clinical biopsy material,buccal swabs, cultured cells, and blood cells. Viral nucleic acid isalso within the scope of the current teachings. In certain embodiments,the target nucleic acid may be present in a double-stranded orsingle-stranded form. The skilled artisan appreciates that gDNA includesnot only full length material, but also fragments generated by anynumber of means, for example but not limited to, enzyme digestion,sonication, shear force, and the like, and that all such material,whether full length or fragmented, represent forms of gDNA that canserve as templates for an amplifying reaction of the current teachings.

A target nucleic acid can be either synthetic or naturally occurring.Certain target nucleic acid, including flanking sequences whereappropriate, can be synthesized using oligonucleotide synthesis methodsthat are well-known in the art. Detailed descriptions of such techniquescan be found in, among other places, Beaucage; and Blackburn and Gait.Automated DNA synthesizers useful for synthesizing target nucleic acidsand other oligonucleotides, including without limitation certain enzymeinhibitors, probes, and primers are commercially available from numeroussources, including for example, the Applied Biosystems DNA SynthesizerModels 381A, 391, 392, and 394 (Applied Biosystems, Foster City,Calif.). Target nucleic acid, including flanking regions whereappropriate, and other oligonucleotides can also be generatedbiosynthetically, using in vivo methodologies and/or in vitromethodologies that are well known in the art. Descriptions of suchtechnologies can be found in, among other places, Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (1989)(hereinafter “Sambrook et al.”); and Ausubel et al., Current Protocolsin Molecular Biology, John Wiley & Sons, Inc., including supplementsthrough Sep. 26, 2005 (hereinafter “Ausubel et al.”).

Target nucleic acids for use in the methods of the current teachings,including but not limited to, gDNA can be obtained from biologicalmaterials using any suitable sample preparation technique known in theart. Commercially available nucleic acid extraction instruments andsystems include, among others, the ABI PRISM® 6100 Nucleic AcidPrepStation and the ABI PRISM® 6700 Nucleic Acid Automated Work Station.Nucleic acid sample preparation reagents and kits are also commerciallyavailable, including without limitation, NucPrep™ Chemistry, BloodPrep™Chemistry, the ABI PRISM® TransPrep System, and PrepMan™ Ultra SamplePreparation Reagent (all from Applied Biosystems); and the miRvana RNAIsolation kit (Ambion, Austin, Tex.). Purified or partially purifiednucleic acid, including without limitation, gDNA and total RNA andtissue-specific nucleic acid preparations, is commercially availablefrom numerous commercial sources, including but not limited to CoriellCell Repositories, Coriell Institute for Medical Research, Camden, N.J.;Serologicals Corp., Norcross, Ga.; Stratagene, La Jolla Calif.; Ambion,Austin, Tex.; and the American Type Culture Collection (ATCC), Manassas,Va.

The terms “amplifying” and “amplification” are used in a broad sense andrefer to any technique known in the art in which a target nucleic acid,an amplicon, at least part of a target nucleic acid, or at least part ofan amplicon, is reproduced or copied (including the synthesis of acomplementary strand or the formation of a ligation probe), typically ina template-dependent manner, including a broad range of techniques foramplifying nucleic acid sequences, either linearly or exponentially.Some amplifying techniques are performed isothermally; someamplification techniques are performed using temperature cycling; someamplification techniques comprise at least one isothermal amplifyingstep and at least one amplifying step comprising thermocycling. Somenon-limiting examples of amplification techniques include primerextension, including without limitation PCR, RT-PCR, asynchronous PCR(A-PCR), asymmetric PCR, quantitative or Q-PCR; ligase chain reaction(LCR), ligase detection reaction (LDR), including without limitationgap-filling and gap oligonucleotide versions of each (see, e.g., Cao,Chapter 1.3 in DNA Amplification: Current Techniques and Applications,Demidov and Broude, eds., Horizon Bioscience (2004; hereinafter “Demidovand Broude”); Abravaya et al., Nucl. Acids Res. 23:675-82 (1995);Lizardi et al., Nat. Genetics 19:225-32 (1998); and Segev, U.S. Pat. No.6,004,826); rolling circle amplification (RCA), sometimes referred to asrolling circle replication (RCR); strand displacement amplification(SDA) and multiple displacement amplification (MDA); nucleic acidstrand-based amplification (NASBA), sometimes referred to astranscription-mediated amplification (TMA) or self-sustained replication(3SR); SPIA™ and RiboSPIA™ amplification (see, e.g., Kurn, U.S. Pat. No.6,251,639 and U.S. Patent Application Publication No. US2003/0017591A1); and helicase-dependent amplification (HDA; see, e.g.,Vincent et al., EMBO Reports 5:795-800 (2004)), and including withoutlimitation multiplex versions and/or combinations thereof, for examplebut not limited to, OLA/PCR, PCR/LDR, PCR/LCR, also known as combinedchain reaction (CCR). Descriptions of certain amplification techniquescan be found in, among other places, Molecular Cloning, A LaboratoryManual, Sambrook and Russell, eds., Cold Spring Harbor Press, 3d ed.(2001; hereinafter “Sambrook and Russell”); Sambrook et al.; Ausubel etal.; PCR Primer; McPherson; Rapley; Lizardi et al., Nat. Genetics19:225-32 (1998); Wiedmann et al., S51-64, in PCR Methods andApplications, Cold Spring Harbor Laboratory Press (1994); Cao, Trends inBiotechnol. 22:38-44 (2004); and Wenz and Schroth, U.S. PatentApplication Publication No. US 2003/0190646A1.

In certain embodiments, amplification techniques comprise at least onecycle of amplification, for example, but not limited to, the steps of:denaturing a double-stranded nucleic acid to separate the componentstrands; hybridizing a primer to a target flanking sequence or aprimer-binding site of an amplicon (or complements of either, asappropriate); and synthesizing a strand of nucleotides in atemplate-dependent manner using a DNA polymerase. In certainembodiments, a cycle of amplification comprises the steps of: denaturinga double-stranded nucleic acid to separate the component strands;hybridizing a first ligation probe and a corresponding second ligationprobe to (1) the target nucleic acid or the complement of the targetnucleic acid or (2) an amplicon; and ligating the adjacently hybridizedprobes with a ligase to form a ligated probe (an exemplary amplicon). Incertain embodiments, a cycle of amplification comprises the steps of:denaturing a double-stranded nucleic acid to separate the componentstrands; hybridizing an upstream cleavage probe and a correspondingdownstream cleavage probe to (1) the target nucleic acid or thecomplement of the target nucleic acid or (2) an amplicon, to form anucleic acid cleavage structure; cleaving the cleavage structure torelease the flap and form a hybridization structure comprising theupstream cleavage probe annealed adjacent to the hybridized fragment ofthe downstream cleavage probe; and optionally ligating the adjacentlyhybridized probes with a ligase to form a ligated probe. The cycle mayor may not be repeated. In certain embodiments, a cycle of amplificationcomprises a multiplicity of amplification cycles, for example but notlimited to 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45cycles or more than 45 cycles of amplification.

In some embodiments, amplifying comprises thermocycling using aninstrument, for example but not limited to, a GeneAmp® PCR System 9700,9600, 2700, or 2400 thermocycler (all from Applied Biosystems). Incertain embodiments, single-stranded amplicons are generated in anamplification reaction, for example but not limited to asymmetric PCR orA-PCR.

Devices have been developed that can perform a thermal cycling reactionand detection with reaction compositions containing a nucleic acid dye,emit a light beam of a specified wavelength, read the intensity of thefluorescent signal emitted from the nucleic acid dye moleculesassociated with double-stranded nucleic acids, and display the intensityof fluorescence after each cycle. Devices comprising a thermal cycler,light beam emitter, and a fluorescent signal detector, have beendescribed, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670,and include, but are not limited to the ABI Prism® 7700 SequenceDetection System (Applied Biosystems, Foster City, Calif.) and the ABIGeneAmp® 5700 Sequence Detection System (Applied Biosystems, FosterCity, Calif.).

In certain embodiments, these functions may be performed by separatedevices. For example but not as a limitation, if one employs a Q-betareplicase reaction for amplification, the reaction may not take place ina thermal cycler, but in a reaction vessel in an instrument that couldinclude a light beam emitted at a specific wavelength, detection of thefluorescent signal, and calculation and display of the amount ofamplification product on a monitor or other read-out device.

In certain embodiments, combined thermal cycling and fluorescencedetecting devices can be used for precise quantification of targetnucleic acid sequences in samples. In certain embodiments, fluorescentsignals can be detected and displayed during and/or after one or morethermal cycles, thus permitting monitoring of amplification products asthe reactions occur in “real time.” In certain embodiments, one can usethe amount of amplification product and number of amplification cyclesto calculate how much of the target nucleic acid sequence was in thesample prior to amplification.

In some embodiments, one ligation probe set is provided for a targetnucleic acid and the target is amplified linearly, for example but notlimited to LDR. In certain embodiments, two ligation probe sets areprovided for a target nucleic acid and the target is amplifiedexponentially, for example but not limited to LCR. In some embodiments,a first cleavage probe and a corresponding second cleavage probe annealwith the target nucleic acid to form a nucleic acid cleavage structurecomprising a overlapping or flap sequence that forms a suitablesubstrate for a cleaving enzyme. In certain embodiments, after cleavage,the first cleavage probe and the hybridized fragment of the secondcleavage probe can be ligated to form a ligated probe. In someembodiments, a ligated probe comprises a primer-binding site and canserve as the template for a primer extension reaction, for example butnot limited to PCR.

Primer extension according to the present teachings is an amplificationprocess comprising elongating a primer that is annealed to a template inthe 5′ to 3′ direction using a DNA polymerase. According to certainembodiments, with appropriate buffers, salts, pH, temperature, andappropriate NTPs (which may, but need not, comprise a nucleotideanalog), a DNA polymerase incorporates nucleotides complementary to thetemplate strand starting at the 3′-end of an annealed primer, togenerate a complementary strand. In certain embodiments, the DNApolymerase used for primer extension lacks or substantially lacks5′-exonuclease activity, 3′-exonuclease activity, or both. In someembodiments, primer extension comprises reverse transcription and theDNA polymerase comprises a reverse transcriptase or a DNA-dependent DNApolymerase that under certain conditions comprises reverse transcriptaseactivity, for example but not limited to, Thermus thermophilus (Tth) DNApolymerase, recombinant Tth DNA polymerase (rTth pol), GeneAmp AccuRTRNA PCR Enzyme, or Thermus species Z05 (TZ05) DNA polymerase (see, e.g.,Smith et al., in PCR Primer, at pages 211-219). In certain embodiments,primer extension comprises a reverse transcriptase and a DNA-dependentDNA polymerase. In certain such embodiments, the reaction compositionmay comprise one DNA polymerase inhibitor or at least two different DNApolymerase inhibitors, for example but not limited to a first DNApolymerase that can form a complex with the reverse transcriptase and asecond DNA polymerase inhibitor that can form a complex with theDNA-dependent DNA polymerase. Descriptions of certain primer extensionreactions can be found in, among other places, Sambrook et al., Sambrookand Russell, Ausubel et al. and Chen et al., U.S. patent applicationSer. No. 10/947,460.

In some embodiments of the current teachings, amplification comprises atwo-step reaction including without limitation a pre-amplification stepwherein a limited number of cycles of amplification occur (for examplebut not limited to 2, 3, 4, or 5 cycles of amplification), then theresulting amplicon is generally diluted and portions of the dilutedamplicon are subjected to additional cycles of amplification in asubsequent amplification step (see, e.g., Marmaro and Gordes, U.S. Pat.No. 6,605,451; and Andersen and Ruff, U.S. Patent ApplicationPublication No. US 2004/0175733). In some embodiments, apre-amplification step, a subsequent amplification step, or both,comprise a DNA polymerase inhibitor.

In certain embodiments, an amplification reaction comprises multiplexamplification, in which a multiplicity of different target nucleic acidsand/or a multiplicity of different amplification product species aresimultaneously amplified using a multiplicity of different primer sets,a multiplicity of different ligation probe sets, a multiplicity ofdifferent cleavage probe sets, or combinations thereof (see, e.g.,Henegariu et al., BioTechniques 23:504-11, 1997; Belgrader et al.,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification (1995); andRapley, particularly in Chapter 79). Certain embodiments of thedisclosed methods comprise a multiplex amplification reaction and asingle-plex amplification reaction, including a multiplicity ofsingle-plex or lower-plexy reactions (for example but not limited to atwo-plex, a three-plex, a four-plex, a five-plex, or a six-plexreaction) performed in parallel.

In certain embodiments, an amplifying reaction comprises asymmetric PCR.According to certain embodiments, asymmetric PCR comprises a reactioncomposition comprising (i) at least one primer pair in which there is anexcess of one primer, relative to the corresponding primer of the primerpair, for example but not limited to a five-fold, a ten-fold, or atwenty-fold excess; (ii) at least one primer pair that comprises only aforward primer or only a reverse primer; (iii) at least one primer pairthat, during given amplification conditions, comprises a primer thatresults in amplification of one strand and a corresponding primer thatis disabled; or (iv) at least one primer pair that meets the descriptionof both (i) and (iii) above. Consequently, when the target nucleic acidand/or amplicon is amplified, an excess of one strand of the subsequentamplification product (relative to its complement) is generated.Descriptions of asymmetric PCR, can be found in, among other places,McPherson, particularly in Chapter 5; and Rapley, particularly inChapter 64.

In certain embodiments, one may use at least one primer pair wherein theTm of one of the primers is higher than the Tm of the other primer,sometimes referred to as A-PCR (see, e.g., Chen et al., U.S. PatentApplication Publication No. US 2003/0207266A1). In certain embodiments,the Tm of the forward primer is at least 4-15° C. different from the Tmof the corresponding reverse primer. In certain embodiments, the Tm ofthe forward primer is at least 8-15° C. different from the Tm of thecorresponding reverse primer. In certain embodiments, the Tm of theforward primer is at least 10-15° C. different from the Tm of thecorresponding reverse primer. In certain embodiments, the Tm of theforward primer is at least 10-12° C. different from the Tm of thecorresponding reverse primer. In certain embodiments, in at least oneprimer pair, the Tm of a forward primer differs from the Tm of thecorresponding reverse primer by at least about 4° C., by at least about8° C., by at least about 10° C., or by at least about 12° C.

In certain embodiments of A-PCR, in addition to the difference in Tm ofthe primers in a primer pair, there is also an excess of one primerrelative to the other primer in the primer pair. In certain embodiments,there is a five- to twenty-fold excess of one primer relative to theother primer in the primer pair. In certain embodiments of A-PCR, theprimer concentration is at least 50 nM.

According to certain A-PCR embodiments, one may use conventional PCR inthe first cycles of amplification such that both primers anneal and bothstrands of a double-stranded amplicon or target nucleic acid areamplified. By raising the temperature in subsequent cycles of the sameamplification reaction, however, one may disable the primer with thelower Tm such that only one strand is amplified. Thus, the subsequentcycles of A-PCR in which the primer with the lower Tm is disabled resultin asymmetric amplification. Consequently, when the target nucleic acidor an amplification product is amplified, an excess of one strand of thesubsequent amplification product (relative to its complement) isgenerated.

According to certain A-PCR embodiments, the level of amplification canbe controlled by changing the number of cycles during the first phase ofconventional PCR cycling. In such embodiments, by changing the number ofinitial conventional cycles, one may vary the amount of thedouble-stranded amplification products that are subjected to thesubsequent cycles of PCR at the higher temperature in which the primerwith the lower Tm is disabled.

Certain methods of optimizing amplification reactions are known to thoseskilled in the art. For example, it is known that PCR may be optimizedby altering times and temperatures for annealing, polymerization, anddenaturing, as well as changing the buffers, salts, and other reagentsin the reaction composition. Optimization may also be affected by thedesign of the probes and/or primers used. For example, the length of theprobes and/or primers, as well as the G-C:A-T ratio may alter theefficiency of annealing, thus altering the amplification reaction.Descriptions of amplification optimization can be found in, among otherplaces, James G. Wetmur, “Nucleic Acid Hybrids, Formation andStructure,” in Molecular Biology and Biotechnology, pp. 605-8, (RobertA. Meyers ed., 1995); McPherson, particularly in Chapter 4; Rapley; andProtocols & Applications Guide, rev. 9/04, Promega.

Certain reaction compositions further comprise dUTP anduracil-N-glycosylase (UNG; e.g., AmpErase®, Applied Biosystems) oruracil-DNA glycosylase (UDG; New England BioLabs, Beverly, Mass.).Discussion of the use of dUTP and UNG in amplification reactions may befound, for example, in Kwok et al., Nature, 339:237-238, 1989;McPherson; Longo et al., Gene, 93:125-128, 1990; and Gelfand et al.,U.S. Pat. No. 5,418,149.

In certain method embodiments, amplification comprises a helicase,including without limitation, E. coli UvrD helicase, DnaB helicase, orbacteriophage T7 gene 4 protein; a DNA polymerase, including withoutlimitation DNA polymerase III or the Klenow fragment of DNA polymeraseI; a helicase accessory protein, including without limitation, MutLprotein; a single-stranded binding protein (SSB), including withoutlimitation, E. coli SSB, T7 gene 2.5 SSB, T4 gene 32 protein, and/orRB49 gene 32 protein; or combinations thereof. In certain embodiments,an enzyme inhibitor comprising a nucleotide sequence and a quencher isdesigned to inhibit the enzymatic activity of a helicase when the enzymeinhibitor and the helicase are associated with each other in a complexat a first temperature, but not at a second temperature, at which theenzyme inhibitor and the helicase have dissociated. In certainembodiments, the nucleotide sequence of a helicase inhibitor comprisesan aptamer. In some embodiments, the nucleotide sequence of a helicaseinhibitor can form a double-stranded segment at the first temperature,but typically not at the second temperature.

In some embodiments, amplification comprises ligase-mediatedamplification techniques, for example but not limited to, LDR, LCR,FEN-LCR, gap oligonucleotide and gap-filling versions of ligationmediated-amplification procedures, padlock versions of ligase-mediatedamplification, and ligation approaches coupled with PCR and/or otheramplification approaches and including multiplex versions thereof (see,e.g., Demidov and Broude, particularly Chapter 1.3; Lizardi et al., Nat.Genetics 19:225-32 (1998); Bi et al., U.S. Pat. No. 6,511,810; and Wenzand Schroth, U.S. Patent Application Publication No. US 2003/0190646A1).According to certain methods comprising ligase-mediated amplification, aligase and a ligase inhibitor that comprises a nucleotide sequence and aquencher associate at a first temperate to form a ligase-ligaseinhibiter complex. When associated with the ligase inhibitor, theenzymatic activity of the ligase is inhibited, which decreases at leastsome of the misligation that could occur in the absence of the ligaseinhibitor, thus decreasing certain secondary amplicons and reducingbackground fluorescence. When the reaction composition comprising theligase-ligase inhibitor complex is heated to a second temperature, theligase inhibitor dissociates from the ligase and adjacently hybridizedprobes can be efficiently ligated. In certain embodiments, the 5′-enddownstream ligation probe and the 3′-end of the corresponding upstreamligation probe are not immediately adjacent when they hybridize to thetarget nucleic acid or its complement, and a gap-filling step isemployed to extend the 3′-end of the upstream probe into juxtapositionwith the 5′-end of the downstream probe. In other embodiments, there isa gap between the 5′-end of the downstream probe and the 3′-end of theupstream probe such that a “gap oligonucleotide” can hybridize in thegap between the opposing ends of the ligation probes. In certain suchembodiments, the 5′-end downstream probe can be ligated to the 3′-end ofthe gap oligonucleotide and the 3′-end of the upstream probe can beligated to the 5′-end of the gap oligonucleotide.

In certain embodiments, the nucleotide sequence of the ligase inhibitorcomprises an aptamer. In some embodiments, the nucleotide sequence of aligase inhibitor can form a double-stranded segment at the firsttemperature, but typically not at the second temperature.

According to certain gap-filling LCR or gap-filling LDR amplificationtechniques, a complex comprising a DNA polymerase and a DNA polymeraseinhibitor can form at a first temperature, inhibiting the DNA polymeraseactivity. In certain embodiments, a ligase and a ligase inhibitor form acomplex at a first temperature to inhibit ligation of mis-annealedligation probes, sometimes referred to as misligation.

Those in the art will appreciate that the disclosed enzyme inhibitors,complexes, methods, and kits can be applied in a variety of differentcontexts in which an enzyme-mediated amplification reaction is performedthat may be subject to mis-annealing of primers and/or probes and thesubsequent formation of undesired secondary amplicons. Anyenzyme-mediated amplification technique that can benefit from the use ofan enzyme inhibitor comprising a quencher to at least decreasebackground fluorescence is within the intended scope of the currentteachings.

An amplified or sequenced target nucleic acid can be detected by anysuitable technique known in the art that comprises measuring,quantitating, and/or observing directly or indirectly, a quenchableemission, including without limitation, fluorescence, chemiluminescence,bioluminescence, phosphorescence, and so forth, for example but notlimited to, laser-induced fluorescence and electrochemiluminescence.According to some embodiments of the disclosed methods, detecting cancomprise any suitable real-time or end-point detection technique. Somenon-limiting examples of suitable detection techniques include meltingcurve analysis, Q-PCR or other real-time technique comprising a nucleicacid dye, and in some embodiments, at least one reporter probe; andelectrophoresis techniques, including without limitation gelelectrophoresis. Those in the art will appreciate that various quenchermoieties are available that collectively cover a broad range ofdetectable emissions and that by pairing a quencher with an appropriateabsorption spectra with an emission source, at least some of theemission from that source can be reduced.

In some embodiments, the methods of the current teachings compriseQ-PCR. The term “quantitative PCR”, or “Q-PCR”, also known as real-timePCR, refers to a variety of methods used to quantify PCR amplificationproducts, either specifically, non-specifically, or both (see, e.g.,Raeymakers, Mol. Biotechnol. 15:115-22 (2000); Joyce, QuantitativeRT-PCR, in Methods in Mol Biol., vol. 193, O'Connell, ed., Humana Press;Pierson et al., Nucl. Acids Res. 31(14):e73 (2003)). Such methodstypically are categorized as kinetics-based systems, that generallydetermine or compare the amplification factor, such as determining thethreshold cycle (C_(t)), or as co-amplification methods, that generallycompare the amount of product generated from simultaneous amplificationof target and standard templates. Q-PCR techniques typically comprisereporter probes, a nucleic acid dye, or both. For example but notlimited to TaqMan® probes (Applied Biosystems), i-probes, molecularbeacons, Eclipse probes, scorpion primers, Lux™ primers, FRET primers,ethidium bromide, and unsymmetrical cyanine dyes, for example but notlimited to, SYBR® Green I (Molecular Probes), YO-PRO-1, Hoechst 33258,BOXTO (TATAA Biocenter, Goteborg, Sweden) and PicoGreen® (MolecularProbes).

In some embodiments, the methods of the current teachings are performedbefore or in conjunction with a sequencing reaction. The term“sequencing” is used in a broad sense herein and refers to any techniqueknown in the art that allows the order of at least some consecutivenucleotides in at least part of a polynucleotide, for example but notlimited to a target nucleic acid or an amplicon, to be identified. Somenon-limiting examples of sequencing techniques include Sanger's dideoxyterminator method and the chemical cleavage method of Maxam and Gilbert,including variations of those methods; sequencing by hybridization;sequencing by synthesis; and restriction mapping. Some sequencingmethods comprise electrophoreses, including capillary electrophoresisand gel electrophoresis; sequencing by hybridization includingmicroarray hybridization; mass spectrometry; and single moleculedetection. In some embodiments, sequencing comprises direct sequencing,duplex sequencing, cycle sequencing, single base extension sequencing(SBE), solid-phase sequencing, or combinations thereof. In someembodiments, sequencing comprises detecting the sequencing product usingan instrument, for example but not limited to an ABI PRISM® 377 DNASequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730xl GeneticAnalyzer, an ABI PRISM® 3700 DNA Analyzer (all from Applied Biosystems),or a mass spectrometer. In some embodiments, sequencing comprisesincorporating a dNTP, including a dATP, a dCTP, a dGTP, a dTTP, a dUTP,a dITP, or combinations thereof and including dideoxyribonucleotideanalogs of dNTPs, into an amplification product.

Those in the art will appreciate that the sequencing method employed isnot typically a limitation of the present methods. Rather any sequencingtechnique that provides the order of at least some consecutivenucleotides of at least part of the corresponding amplicon or targetnucleic acid can typically be used with the current methods. In someembodiments, unincorporated primers and/or dNTPs are removed prior to asequencing step by enzymatic degradation, including without limitationexonuclease I and shrimp alkaline phosphatase digestion, for example butnot limited to the ExoSAP-IT® reagent (USB Corp., Cleveland, Ohio). Insome embodiments, unincorporated primers, dNTPs, and/or ddNTPs areremoved by gel or column purification, sedimentation, filtration, beads,magnetic separation, or hybridization-based pull out, as appropriate(see, e.g., ABI PRISM® Duplex™ 384 Well F/R Sequence Capture Kit,Applied Biosystems P/N 4308082). In certain embodiments, a reactioncomposition comprising an amplification product, or at least part ofsuch a reaction composition, is subjected to a sequencing reactionwithout an intervening purification step (see, e.g., Baskin et al., U.S.Patent Application Publication No. US 2002/0137047 A1). Descriptions ofsequencing techniques can be found in, among other places, McPherson,particularly in Chapter 5; Sambrook and Russell; Ausubel et al.;Siuzdak, The Expanding Role of Mass Spectrometry in Biotechnology, MCCPress, 2003, particularly in Chapter 7; and Rapley, particularly in PartVI.

In some embodiments, the disclosed methods and kits comprise amicrofluidics device, “lab on a chip”, or micrototal analytical system(μTAS). In some embodiments, sample preparation is performed using amicrofluidics device. In some embodiments, an amplification reaction isperformed using a microfluidics device. In some embodiments, asequencing or real-time PCR reaction is performed using a microfluidicdevice. In some embodiments, the nucleotide sequence of at least a partof an amplification product is obtained using a microfluidics device.Descriptions of exemplary microfluidic devices can be found in, amongother places, Published PCT Application Nos. WO/0185341 and WO04/011666; Kartalov and Quake, Nucl. Acids Res. 32:2873-79, 2004; andFiorini and Chiu, BioTechniques 38:429-46, 2005.

Certain Exemplary Embodiments

The present teachings provide compositions, methods, and kits foramplifying a target nucleic acid and for decreasing backgroundfluorescence, typically in a reaction composition comprising at leastone enzyme and at least one enzyme inhibitor that includes at least onenucleotide sequence and at least one quencher.

The instant enzyme inhibitors comprise a nucleotide sequence and aquencher. The nucleotide sequence of such enzyme inhibitors are designedto decrease the formation of undesired amplification products,particularly due to mispriming events at non-target sequences,mis-annealing of ligation and/or cleavage probes, and primer dimerformation, by inhibiting enzyme activity at a first temperature, but notat a second temperature. The decreased level of secondary ampliconformation reduces at least one component of non-specific fluorescence inthe reaction composition. The disclosed enzyme inhibitors are alsodesigned to be self-quenching under appropriate conditions. The quenchermoiety of the disclosed inhibitors are designed to absorb at least someof the fluorescent signal generated by the association of nucleic aciddye molecules with double-stranded segment(s) of the enzyme inhibitor atthe first temperature range, either when the enzyme inhibitor is free insolution or complexed with an enzyme. Thus, the quencher of the enzymeinhibitor reduces at least some of this second source of backgroundfluorescence, further decreasing the non-specific fluorescence in thereaction composition.

Certain Exemplary Enzyme Inhibitors

According to the current teachings, enzyme inhibitors comprising anucleotide sequence and a quencher are designed to inhibit at least oneenzymatic activity of an enzyme while the enzyme inhibitor is associatedwith the enzyme in an enzyme inhibitor-enzyme complex. The nucleotidesequence of the enzyme inhibitors are designed so that they can form astructure comprising at least one double-stranded segment and thequencher(s) are selected to be able to absorb at least some of thefluorescence emitted from a nucleic acid dye when associated with thedouble-stranded segment of the enzyme inhibitor. The enzyme-enzymeinhibitor complexes can form and/or remain associated at a firsttemperature, for example but not limited to, room temperature (typicallyabout 22° C.-28° C.) and temperatures below, at, or slightly above thedesired template extension temperature. When a reaction compositioncomprising an enzyme-enzyme inhibitor complex is heated to a secondtemperature, the enzyme is released as the complex dissociates. Thedisclosed RNA polymerase inhibitors are designed to inhibit thepolymerization activity of an RNA polymerase when the inhibitor and theRNA polymerase are associated in a complex. The disclosed ligaseinhibitors are designed to inhibit the formation of a phosphodiesterbetween two adjacently hybridized nucleotide strands on a template whenthe ligase inhibitor and the ligase are associated in a complex,including the ligation of mis-annealed ligation probes. The disclosedhelicase inhibitors are designed to inhibit the helicase's ability tocatalyze the unwinding of double-stranded nucleic acids when thehelicase inhibitor and the helicase are associated in a complex. Certaindisclosed cleaving enzyme inhibitors are designed to inhibit the5′-nuclease activity of the cleaving enzyme when the cleaving enzymeinhibitor and the cleaving enzyme are associated in a complex. Incertain embodiments, the nucleotide sequence of a ligase inhibitor, anRNA polymerase inhibitor, a helicase inhibitor, and/or a cleaving enzymeinhibitor comprises an aptamer. The inhibitory ability of the enzymeinhibitors of the current teachings are typically not significantlydependent on the exact sequence of the inhibitor. Rather, the overallstructure of the enzyme inhibitor and its melting temperature are themajor determinants of whether an enzyme inhibitor will inhibit theintended enzymatic activity of the corresponding enzyme. In certainembodiments, an enzyme inhibitor is designed to assume a conformation ata first temperature that mimics the substrate of the correspondingenzyme, allowing the enzyme to associate with the inhibitor to form acomplex in which the enzymatic activity of the enzyme is inhibited. At asecond temperature, the conformation of the enzyme inhibitor can changeso that it no longer mimics the substrate and the enzyme is releasedfrom the complex. Thus, the disclosed inhibitors typically exhibitsignificantly less, if any, inhibitory effect when they aresubstantially single-stranded and/or not in a complex with the enzyme.In some embodiments, the nucleotide sequence of an enzyme inhibitorcomprises a deoxyribonucleotide, a ribonucleotide, a nucleotide analog,a non-nucleotide linker, or combinations thereof.

The disclosed ligase inhibitors do not significantly interfere with theannealing of ligation probes or cleavage probes to correspondingsequences on a target nucleic acid or a desired amplicon, for examplebut not limited to a ligated probe. The disclosed helicase inhibitors donot significantly interfere with the hybridization of primers tocorresponding target flanking sequences or amplicons. The disclosedcleaving enzyme inhibitors do not significantly interfere with theannealing or cleavage probes or ligation probes to correspondingsequences on a target nucleic acid or desired amplicon or thehybridization of primers with corresponding target flanking sequencesand/or amplicons.

The disclosed DNA polymerase inhibitors are designed to inhibit thepolymerization activity of a DNA polymerase when the inhibitor isassociated with the DNA polymerase, and optionally a NTP and/or anucleotide analog, in a DNA polymerase inhibitor-DNA polymerase complexat a first temperature, for example but not limited to, temperaturesapproximately the same as or below the Tm of the primer. The inhibitoryability of the DNA polymerase inhibitor of the current teachings isgenerally not significantly dependent on the exact sequence of theinhibitor. Rather, the overall structure of the DNA polymerase inhibitorand its melting temperature are the major determinants of whether a DNApolymerase inhibitor will inhibit the enzymatic activity of the DNApolymerase, i.e., polymerization. Typically, the disclosed DNApolymerase inhibitors will interfere with the polymerization activity ofthe DNA polymerase when they comprise a double-stranded segment and areassociated with the DNA polymerase, and optionally a NTP and/or anucleotide analog, in a complex. The disclosed DNA polymeraseinhibitors, however, exhibit substantially less, if any, inhibitoryeffect when they are single-stranded and not in a complex with the DNApolymerase. In certain embodiments, the Tm of the DNA polymeraseinhibitors is selected to be approximately the same as or lower than thetemperature used for primer extension of the annealed primers employedin the selected polymerization or primer extension reaction, but notalways. In some embodiments, the melting temperatures of the DNApolymerase inhibitors are somewhat above the primer extensiontemperature, for example but not limited to reaction compositionswherein the DNA polymerase inhibitors are used at low concentrations.

Typically, a DNA polymerase inhibitor of the current teachings comprisesat least one double-stranded segment at or below the first temperature,but is single-stranded or substantially single-stranded at or above thesecond temperature. Thus at a first temperature, the enzymatic activityof the DNA polymerase in a complex is inhibited, while at the secondtemperature, the DNA polymerase is active and amplification reactionscan occur.

Exemplary first temperatures include 22° C., 23° C., 24° C., 25° C., 26°C., 27° C., 28° C., 29° C., 30° C., about 22° C. to about 40° C., about25° C. to about 35° C., and about 22° C. to about 28° C., and expresslyincluding all intervening temperatures in the specified firsttemperature ranges. Exemplary second temperatures include: 42° C., 43°C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52°C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61°C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70°C., 71° C., 72° C., 73° C., about 48° C. to about 73° C., about 53° C.to about 67° C., about 63° C. to about 67° C., and about 64° C. to about66° C., and expressly including all intervening temperatures in thespecified second temperature ranges. Those in the art will understandthat the appropriate first and second temperatures for a givenamplification reaction will depend, at least in part, on the enzyme, theTm of the enzyme inhibitor, and/or the Tm of the primer(s) and/orprobes, but that appropriate temperatures can be routinely determined,without undue experimentation, using methods known in the art andinformed by the current teachings.

In certain embodiments, the nucleotide sequence of a DNA polymeraseinhibitor of the present teachings comprises a single oligonucleotide.In some embodiments, such DNA polymerase inhibitors comprise a firstregion, a second region, a third region, and optionally, a fourthregion; and the first region is complementary to the third region. Underappropriate conditions, including at a first temperature, the firstregion and the third region of such DNA polymerase inhibitors can annealand form at least one double-stranded segment so that the DNA polymeraseinhibitor assumes a stem-loop or hairpin conformation. In certainembodiments, only a subset of nucleotides in the first region arecomplementary with the corresponding subset of nucleotides in the thirdregion. In some embodiments, the disclosed DNA polymerase inhibitorscomprise a nucleotide analog that may or may not affect the Tm of theDNA polymerase inhibitor.

Some exemplary DNA polymerase inhibitors comprising one oligonucleotideare depicted schematically in FIG. 1. The illustrative DNA polymeraseinhibitor shown in FIG. 1A comprises a first region (1) shown with blackstripes throughout FIG. 1, a second region (2) shown with a wavy linethroughout FIG. 1, a third region (3), and an optional fourth region([4], shown in brackets to indicate that it is optional in thisembodiment) shown shaded in black throughout FIG. 1. The 3′-end of thisexemplary inhibitor is non-extendable due to the terminal nucleotidecomprising a dideoxycytosine (shown as ddC). The first region (1)further comprises a quencher (5). The exemplary inhibitor is shown withthe first region (1) annealed to the third region (3) to form adouble-stranded segment, so that the inhibitor is in a stem-loopconformation with the second region (2) forming the loop and the fourthregion (4) as a 5′ single-stranded overhang. In certain embodiments, thesingle-stranded overhang of the fourth region of such a DNA polymeraseinhibitor comprises at least some ribonucleotides, particularly when theinhibitor is designed to complex with certain reverse transcriptases.The exemplary DNA polymerase inhibitor depicted in FIG. 1B comprises afirst region (1), a second region (2), and a third region (3), but not afourth region. The first region (1) and third region (3) are shownannealed to form a stem and the second region (2) forming a loopstructure and further comprising a quencher (5). The illustrative DNApolymerase inhibitor shown in FIG. 1C comprises a first region (1)comprising a first quencher (6), shown as Q1, a second region (2)comprising a second quencher (7), shown as Q2, a third region (3), andan optional fourth region ([4]). The exemplary DNA polymerase inhibitorshown in FIG. 1D comprises a first region (1), a second region (2), athird region (3), and an optional fourth region ([4]) that comprises aquencher (5) at the 5′-end, shown as Q.

In certain DNA polymerase inhibitor embodiments, the nucleotide sequencecomprises a first region, a second region, a third region, a fourthregion, a fifth region, and a sixth region; wherein the first region iscomplementary with the third region and the first region and the thirdregion can form at least one double-stranded segment at a firsttemperature; wherein the fourth region is complementary with the sixthregion and the fourth region and the sixth region can form at least onedouble-stranded segment at a first temperature; wherein there is atleast one single-stranded region between the 3′-end of the sixth regionand the 5′-end of the first region; and wherein the 3′-end of the sixthregion comprises a non-extendible nucleotide.

In other DNA polymerase inhibitor embodiments, the nucleotide sequencecomprises at least two different oligonucleotides, for example but notlimited to, a first oligonucleotide and a second oligonucleotide. Incertain embodiments wherein the DNA polymerase inhibitor comprises twooligonucleotides, the first oligonucleotide comprises a first region andthe second oligonucleotide comprises a third region and optionally, afourth region, and the first region of the first oligonucleotide iscomplementary to the third region of the second oligonucleotide. Incertain embodiments, only a subset of nucleotides in the first region iscomplementary with the corresponding segment(s) of the third region.Under appropriate conditions, including at a first temperature, thefirst region of the first oligonucleotide and the third region of thesecond oligonucleotide can anneal to form a duplex comprising at leastone double-stranded segment. When the DNA polymerase inhibitors of thecurrent teachings are heated to a second temperature, for example butnot limited to in a second temperature range, they assume asingle-stranded or substantially single-stranded conformation, not astem-loop or a duplex conformation.

Some illustrative enzyme inhibitors comprising two or moreoligonucleotides are depicted schematically in FIG. 2. The exemplary DNApolymerase inhibitor shown in FIG. 2A comprises a first oligonucleotidecomprising first region (1) shown with black stripes throughout FIG. 2,annealed to a second oligonucleotide that comprises a third region (3)and a fourth region (4) shown shaded in black throughout FIG. 2. Thefirst oligonucleotide of this exemplary DNA polymerase inhibitor furthercomprises a quencher, shown as Q. The exemplary DNA polymerase inhibitordepicted in FIG. 2B comprises a first oligonucleotide comprising a firstregion (1), annealed to a second oligonucleotide comprising a thirdregion (3) and a fourth region (4). In this illustrative DNA polymeraseinhibitor, the quencher (Q) is shown attached to the fourth region (4).The illustrative DNA polymerase inhibitor shown in FIG. 2C comprises afirst oligonucleotide comprising a first region (1) comprising a firstquencher (shown as Q1) and a second oligonucleotide comprising a thirdregion (3), and a fourth region (4) comprising a second quencher (shownas Q2). The exemplary DNA polymerase inhibitor shown in FIG. 2Dcomprises a first oligonucleotide comprising a first region (1) annealedto a second oligonucleotide comprising a third region (3), wherein thesecond oligonucleotide comprises a quencher (shown as Q). Theillustrative DNA polymerase inhibitor shown in FIG. 2E comprises a firstoligonucleotide comprising a first region (1) and annealed to a secondoligonucleotide comprising a third region (3), wherein both the firstoligonucleotide and the second oligonucleotide comprise a quencher(shown as Q1 and Q2).

In certain embodiments, the nucleotide sequence of the DNA polymeraseinhibitor comprises an aptamer that binds to and inhibits the enzymaticactivity of the DNA polymerase when bound by the aptamer. In someembodiments, a DNA polymerase inhibitor comprises an aptamer thatcomprises at least one double-stranded segment. When the aptamer is freein solution or is bound to the DNA polymerase in a complex, the quencherabsorbs at least some of the fluorescent signal generated by nucleicacid dye molecules associated with the aptamer.

The disclosed DNA polymerase inhibitors do not significantly interferewith primer hybridization with corresponding target flanking sequencesand/or amplicons. In addition to decreasing the fluorescent intensity ofthe nucleic acid dye molecules associated with the double-strandedsegment of DNA polymerase inhibitors and decreasing formation ofsecondary amplicons, some DNA polymerase inhibitors of the currentteachings increase the yield of desired amplicons relative to parallelamplification reactions not comprising the DNA polymerase inhibitors.

In some embodiments, the 3′-end of a nucleotide sequence of a DNApolymerase inhibitor is not extendible by a DNA polymerase, typicallydue to the presence of a non-extendible nucleotide, including withoutlimitation a terminal nucleotide comprising a blocking group. A blockinggroup is a chemical moiety that can be added to a nucleotide or anucleic acid to prevent or minimize nucleotide addition by a DNApolymerase. By adding a blocking group to the terminal 3′-OH, thenucleotide is no longer able to participate in phosphodiester bondformation catalyzed by the DNA polymerase. Some non-limiting examples ofblocking groups include an alkyl group, non-nucleotide linkers,phosphorothioate, alkane-diol residues, PNA, LNA, nucleotide analogscomprising 3′ amino groups in place of the 3′-hydroxyl group, nucleotideanalogs comprising 5′ hydroxyl groups in place of the 5′ phosphategroup, and nucleotide derivatives lacking a 3′ OH group. An alkylblocking group is a saturated hydrocarbon that can be straight chained,branched, cyclic, or combinations thereof. Some non-limiting examples ofnon-extendable nucleotides include nucleotides that have a 3′-hydroxylgroup that has been modified such as by substitution with hydrogen orfluorine or by formation of an ester, amide, sulfate or glycoside. Thesenucleotides are generally not chain extendable. Other examples ofnon-extendable nucleotides that can be used include nucleotides thathave modified ribose moieties. In certain embodiments, ribonucleotidesmay serve as non-extendable nucleotides because oligonucleotidesterminating in ribonucleotides cannot be extended by certain DNApolymerases. The ribose can be modified to include 3′-deoxy derivativesincluding those in which the 3′-hydroxy is replaced by a functionalgroup other than hydrogen, for example, as an azide group. In certainembodiments, a non-extendible nucleotide comprises a dideoxynucleotide(ddN), for example but not limited to, a dideoxyadenosine (ddA), adideoxycytosine (ddC), a dideoxyguanosine (ddG), a dideoxythymidine(ddT), or a dideoxyuridine (ddU).

In some embodiments, an enzyme inhibitor comprises two quenchers, threequenchers, or more than three quenchers. In certain inhibitorembodiments, a first region comprises a quencher and/or a third regioncomprises a third quencher. In certain embodiments, a second regioncomprises a quencher. In some embodiments, a fourth region comprises aquencher. In certain embodiments, a fifth region comprises a quencher.In certain embodiments, a sixth region comprises a quencher. In someembodiments, an enzyme inhibitor comprises a quencher at the 3′-end ofthe nucleotide sequence, the 5′-end of the nucleotide sequence, and/orinternally. In some embodiments, an enzyme inhibitor comprises a secondregion and in some embodiments a fifth region that forms the loop of astem-loop conformation. In certain embodiments, a loop comprises aquencher.

The disclosed ligase inhibitors do not significantly interfere withligation probe annealing, and in certain embodiments, cleavage probeannealing and/or primer annealing, with corresponding target nucleicacids and/or amplicons. The disclosed cleaving enzyme inhibitors do notsignificantly interfere with cleavage probe annealing, and in certainembodiments, ligation probe annealing and/or primer annealing, withcorresponding target nucleic acids or amplicons. The disclosed helicaseinhibitors do not significantly interfere with primer annealing, and incertain embodiments, cleavage probe and/or ligation probe annealing,with corresponding target nucleic acids and/or amplicons. In addition todecreasing the fluorescent intensity of the nucleic acid dye moleculesassociated with the double-stranded segment of enzyme inhibitors anddecreasing formation of secondary amplicons, some enzyme inhibitors ofthe current teachings may increase the yield of desired ampliconsrelative to parallel amplification reactions not comprising the enzymeinhibitors.

In certain embodiments, a double-stranded segment of an enzyme inhibitorcomprises an internal base pair mismatch. In certain embodiments, anenzyme inhibitor comprises a loop structure, typically stem-loopstructures comprising a double-stranded segment and a single-strandedloop. In certain embodiments, an enzyme inhibitor comprises two loopstructures. In some embodiments, a second region and/or a fifth regionof an enzyme inhibitor can form a loop structure at a first temperaturewhen complementary sequences of the inhibitor anneal with each other,for example but not limited to the first region annealing with the thirdregion; and/or the fourth region annealing with the sixth region. Incertain embodiments, the second region, a fifth region, or a secondregion and a fifth region of the nucleotide sequence comprises 2-12nucleotides and/or nucleotide analogs, and in some embodiments, 2-6nucleotides and/or nucleotide analogs. In some embodiments, the secondand/or fifth region comprises a non-nucleotide linker. In certainembodiments, the second region, the fifth region, or the second and thefifth region of an enzyme inhibitor consists of, consists essentiallyof, or comprises the sequence (T)n, wherein n is any number of Tnucleotides between 1 and 8, for example but not limited to, TT, TTT,TTTT, or TTTTT. In other embodiments, the second region and/or the fifthregion, consists of, consists essentially of, or comprises thenucleotides A, C, and/or G, including without limitation nucleotideanalogs of any of these. In some embodiments, the second region and/orthe fifth region comprises (1) at least one nucleotide analog, forexample but not limited to a PNA and/or an LNA and/or (2) anon-nucleotide linker, for example but not limited to a non-nucleotidecomprising a hydrocarbon group (—CH₂—), including without limitation,linkers comprising an alkane, alkene, or alkyne portion, and ethyleneglycol, including without limitation polyethylene glycol (PEG).Typically the linker group is not hydrophobic. In certain embodiments, alinker is hydrophilic or at least portions of the linker havehydrophilic properties. Those in the art will appreciate that thecomposition of a linker in the disclosed enzyme inhibitors is generallynot a limitation, provided that the linker does not interfere with theenzyme-enzyme inhibitor interaction and that the linker is sufficientlyflexible to allow the enzyme inhibitor to self anneal at the firsttemperature.

In some embodiments, a DNA polymerase inhibitor comprises a minor groovebinder on the 3′-end, the 5′-end, or both the 3′-end and the 5-end ofthe nucleotide sequence. In some embodiments, the minor groove binder islocated internally. In certain embodiments, the minor groove binderfurther comprises a quencher, for example but not limited to, a MGB-NFQ(Applied Biosystems). Non-limiting examples of minor groove bindersinclude, antibiotics such as netropsin, distamycin, berenil, pentamidineand other aromatic diamidines, Hoechst 33258, SN 6999, aureolicanti-tumor drugs such as chromomycin and mithramycin, CC-1065,dihydrocyclopyrroloindole tripeptide (DPI₃),1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃), and relatedcompounds and analogs. Descriptions of minor groove binders can be foundin, among other places, Nucleic Acids in Chemistry and Biology, 2d ed.,Blackburn and Gait, particularly in section 8.3; Kumar et al., Nucl.Acids Res. 26:831-38, 1998; Kutyavin et al., Nucl. Acids Res. 28:655-61,2000; Turner and Denny, Curr. Drug Targets 1:1-14, 2000; Kutyavin etal., Nucl. Acids Res. 25:3718-25, 1997; Lukhtanov et al., Bioconjug.Chem. 7:564-7, 1996; Lukhtanov et al., Bioconjug. Chem. 6: 418-26, 1995;U.S. Pat. No. 6,426,408; and PCT Published Application No. WO 03/078450.Those in the art understand that minor groove binders typically increasethe T_(m) of the oligonucleotide to which they are attached, allowingsuch oligonucleotides to effectively hybridize at higher temperatures.Minor groove binders are commercially available from, among othersources, Applied Biosystems (Foster City, Calif.) and Epoch Biosciences(Bothell, Wash.).

In some embodiments, the nucleotide sequence of an enzyme inhibitorcomprises a universal base. In some embodiments, a DNA polymeraseinhibitor includes a fourth region or a sixth region that comprises auniversal base. In certain embodiments, the nucleotide of the fourthregion that is immediately adjacent to the third region of the DNApolymerase inhibitor comprises a universal base. In certain embodiments,the nucleotide of the sixth region that is immediately adjacent to thesingle-stranded region between the sixth region and the first region ofthe DNA polymerase inhibitor comprises a universal base. In someembodiments, the universal base interacts with a NTP in a DNA polymeraseinhibitor-DNA polymerase complex.

Those in the art will appreciate that the Tm of an enzyme inhibitor canbe determined empirically, using well-known methods and instructed bythe current teachings, and without undue experimentation; or the Tm canbe estimated using algorithms. Several formulas and computer algorithmsfor calculating an estimated Tm, including chimeric oligomers comprisingconventional nucleotides and/or nucleotide analogs, are well-known inthe art. According to one such predictive formula for oligonucleotides,Tm=(4× number of G+C)+(2× number of A+T). The Tm for a particularoligonucleotide, such as an enzyme inhibitor, a probe, or a primer, canalso be routinely determined using known methods, without undueexperimentation. Descriptions of Tm/melting temperatures and theircalculation can be found in, among other places, Rapley; Nielsen, ExiqonTechnical Note LNA 02/07.2002, Exiqon A/S; McPherson; Finn et al., Nucl.Acids Res. 17:3357-63, 1996.

The melting temperature of the enzyme inhibitors of the currentteachings can be modulated in a variety of ways. For example, those inthe art understand that the length and/or composition of thecomplementary sequences of the first and third regions, and in certainembodiments, the fourth and sixth regions, can be varied to increase ordecrease the melting temperature of an enzyme inhibitor; in certaininhibitor embodiments, the length and/or composition of thecomplementary sequences of the fourth and sixth regions can be varied toincrease or decrease the Tm of the enzyme inhibitor. Hence, in general,a double-stranded segment with greater numbers of hybridizing base pairswill usually melt at higher temperatures than a double-stranded segmentwith lesser numbers of hybridizing base pairs. However, if a longdouble-stranded segment is desired, one of skill in the art canintroduce base pair mismatches, for example but not limited to, G:T basepairs, to modulate the melting temperature. In certain embodiments, adouble-stranded segment of an enzyme inhibitor comprises one mismatchedbase pair, two mismatched base pairs, three mismatched base pairs, fourmismatched base pairs, or more than four mismatched base pairs, whereintwo or more mismatched base pairs can, but need not be, contiguous.

Therefore, the double-stranded segment of the disclosed enzymeinhibitors need not be 100% complementary. Instead, a double-strandedsegment can have a number, or a certain percentage, of mismatches orwobble base pairs. For example, the double-stranded segment can haveabout 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, or about 20% base pairmismatches. In certain embodiments, the melting temperature of an enzymeinhibitor is modulated by designing a double-stranded segment thatcomprises an abasic nucleotide analog, for example but not limited to ananalog comprising a sugar or a sugar analog and a phosphate or aphosphate analog, but not a nucleotide base or a nucleotide base analog,which among other things, eliminates a base pair in the double-strandedregion.

The melting temperature of an enzyme inhibitor comprising a secondregion and/or a fifth region can also be modulated by increasing ordecreasing the number of nucleotides and/or nucleotide analogs in theloop. The melting temperature of an enzyme inhibitor comprising a singleoligonucleotide can also be modulated by the presence or absence of oneor more “GC clamp” at the junction between a region that can comprise atleast one double-stranded segment at a first temperature and a regionthat does not comprise a double-stranded segment at the firsttemperature. For example but not limited to the base of a loopstructure, including without limitation, in certain embodiments, thenucleotide(s) of the first region that are adjacent to the second regionand that anneal with the nucleotide(s) of the third region that areadjacent to the second region (see, e.g., FIG. 1A), and/or in someembodiments, the nucleotide(s) of the fourth region that are adjacent tothe fifth region and that anneal with the nucleotide(s) of the sixthregion that are adjacent to the fifth region (see, e.g., FIG. 1 E).Likewise, the Tm of enzyme inhibitors comprising two or moreoligonucleotides can be modulated by the presence or absence of GCclamps, particularly when they are located at one or both ends ofcomplementary segments of the first and third regions of the enzymeinhibitors, including without limitation, the base of a loop, ifappropriate; and in certain embodiments, at one of both ends ofcomplementary segments of the fourth and sixth regions of the enzymeinhibitors. The melting temperature of an enzyme inhibitor can also bemodulated by nucleotide analogs in the first and/or third regions of thenucleotide sequence, and in certain embodiments, the fourth and/or sixthregions of the nucleotide sequence, for example but not limited todeaza-dA. Some non-limiting examples of nucleotide analogs that increasethe Tm include C-5 propynyl-dC or 5-methyl-2′-deoxycytidine substitutedfor dC; 2,6-diaminopurine 2′-deoxyriboside (2-amino-dA) substituted fordA; and C-5 propynyl-dU for dT; which increase the relative meltingtemperature approximately 2.8° C., 1.3° C., 3.0° C., and 1.7° C. persubstitution, respectively.

When considering the length of the double-stranded segment(s), themelting temperature of the enzyme inhibitor should be considered. Forexample but not as a limitation, if the T_(m) of a DNA polymeraseinhibitor is too high, it may denature at a temperature above thetemperature used in the amplification for primer extension, therebycausing inhibition of the desired polymerization reaction and adecreased yield of the desired amplicon. If the T_(m) is too low, theDNA polymerase inhibitor may melt and become inactive at temperaturesthat permit the primers to hybridize to non-target nucleic acids and beextended. When the DNA polymerase is able to amplify such non-targetnucleic acids, many undesirable products will be present in the finalamplified product mixture, including primer-dimers. In certainembodiments, an enzyme inhibitor has a melting temperature that is closeto, but not significantly greater than, the selected extension.ligation, and/or cleavage reaction temperature of the amplificationreaction, as appropriate. In some embodiments, particularly when theenzyme inhibitors are used at low concentrations, enzyme inhibitors withmelting temperatures above the primer extension temperature, theligation temperature, or the cleavage reaction temperature, asappropriate, can be used. Typically, one of skill in the art candetermine the melting temperature of an enzyme inhibitor under theconditions in which it will be used, for example, under nucleic acidpolymerization conditions.

An exemplary DNA polymerase inhibitor comprises, consists of, orconsists essentially of:

(SEQ ID NO: 3) 5′-[TCTGG]GATA(deazadA)TT(deazadA)TGGTA (deazadA)ATATGT(DABCYL-T)TT C(deazadA)TATTTATT (deazadA)TA(deazadA)TTATC(MGB-NFQ)-3′,wherein the fourth region is shown in brackets, the third region isshown underlined, the second region is shown in bold, and the firstregion is shown in italics, and wherein the second region comprises afirst quencher (shown as DABCYL in this example) and the first regioncomprises a minor groove binder comprising a second quencher (shown asMGB-NFQ in this example). The first region is substantiallycomplementary to the third region due to the two internal G:T base pairmismatches between the two regions but the DNA polymerase inhibitor isstill self-annealing at a first temperature. In some embodiments of thisillustrative DNA polymerase inhibitor, the terminal C nucleotide on the3′-end of the DNA polymerase inhibitor comprises the nucleotide analogdideoxycytosine (ddC). In some embodiments, the second region comprises,consists of, or consists essentially of TT, TTT, or TTTTT. In otherembodiments, the second region comprises a non-nucleotide linker. Insome embodiments, the second region does not comprise a quencher. Incertain embodiments, the 5′-end of the DNA polymerase inhibitor furthercomprises a quencher. In certain embodiments, at least one of the Gnucleotides of the DNA polymerase inhibitor comprises the nucleotideanalog deaza-dG. In some embodiments, the first quencher comprises: aTAMRA™ (carboxytetramethylrhodamine); a Black Hole Quencher dye, forexample but not limited to BHQ-1, BHQ-2, or BHQ-3 (BiosearchTechnologies, Inc.); an OREGON GREEN® dye (Molecular Probes); a ROX™(carboxy-X-rhodamine); a DABSYL (4-dimethylaminoazobenzene-4′-sulfonylchloride); or a TET (tetrachlorofluorescein), instead of or in additionto the DABCYL moiety. In some embodiments, the second quencher comprisesa DABSYL, a DABCYL, a TAMRA, a Black Hole Quencher, a ROX, an OREGONGREEN, or a TET, instead of or in addition to the MGB-NFQ. The choice ofquencher(s) is typically not a limitation of the current teachingsprovided that the selected quencher(s) can absorb fluorescence at thewavelength that is characteristic of the nucleic acid dye and that thequencher and/or the location of the quencher in the inhibitor does notsubstantially decrease the ability of the inhibitor to self-annealand/or complex with the enzyme.

Another exemplary DNA polymerase inhibitor comprises, consists of, orconsists essentially of

(SEQ ID NO: 2) 5′-(TET)-[TTCTGG]GATAATTATGGTAAATATAT TTT ATATATTTATTATAATTATddC-3′,wherein the fourth region is shown in brackets, the third region isshown underlined, the second region is shown in bold, and the firstregion is shown in italics, and wherein the fourth region comprises aquencher (shown as TET in this example). The first region iscomplementary to the third region. The terminal C nucleotide on the3′-end of the first region of the DNA polymerase inhibitor comprises thenucleotide analog dideoxycytosine (ddC), rendering this illustrative DNApolymerase inhibitor non-extendible. In some embodiments, the secondregion comprises, consists of, or consists essentially of TT, TTTT, orTTTTT. In some embodiments, the second region comprises a non-nucleotidelinker. In certain embodiments, the second region does not comprise aquencher. In certain embodiments, the 5′-end of the DNA polymeraseinhibitor further comprises a quencher. In certain embodiments, at leastone of the G nucleotides comprises the nucleotide analog deaza-dG, atleast one A nucleotide comprises the nucleotide analog deaza-dA, or atleast one of the G nucleotides comprises a deaza-dG and at least one Anucleotide a deaza-dA. In some embodiments, the quencher comprises aTAMRA, a Black Hole Quencher dye, a ROX, an OREGON GREEN, a DABCYL, or aDABSYL instead of or in addition to the TET moiety.

Those in the art will appreciate that typically the length andnucleotide and/or nucleotide analog composition of the disclosed enzymeinhibitors can be varied to optimize the stability of the inhibitor,particularly the double-stranded segment(s) and to increase its abilityto inhibit the enzymatic activity of the corresponding enzyme whenassociated in a complex. Those in the art will also appreciate that thedisclosed enzyme inhibitors are typically more effective in inhibitingthe formation of secondary amplification products when the dissociationrate, sometimes referred to as the “off-rate”, of the enzyme-enzymeinhibitor complex at the first temperature is slow. However, in certainapplications, one may be able to compensate for “faster” off-rates byusing higher concentrations of the enzyme inhibitor. Those in the artwill understand that an appropriate concentration of enzyme inhibitorfor a particular application can be determined empirically.

The enzyme inhibitors of the current teachings are particularly usefulwhen detecting comprises a melting curve analysis, sometimes referred toas dissociation curve analysis. To generate a melting or dissociationcurve, the reaction composition is heated, typically in a step-wise orincremental fashion, and the fluorescence of the reaction mixture isdetected at appropriate intervals. Initially, the non-specificfluorescence in the reaction composition is reduced during the initialheating process due to the quencher moiety in the enzyme inhibitor,which reduces the fluorescence emitted from the nucleic acid dyemolecules associated with the double-stranded segment(s) of the enzymeinhibitor in the first temperature range. As the temperature increasesto the second temperature, the double-stranded segment(s) of the enzymeinhibitor begin to melt, releasing the nucleic acid dye molecules thathad been associated with the double-stranded segments of the enzymeinhibitor. A peak in the dissociation curve (plotted as the firstderivative of the fluorescence versus temperature) would be expected toappear due to the enzyme inhibitor dissociating which could complicatethe evaluation of one or more amplicons. Due to the presence of thequencher in the enzyme inhibitor, the dissociation peak associated withthe melting of the inhibitor is decreased or not detected because thequencher absorbs at least some of the fluorescence emitted from theassociated dye molecules, which at least diminishes the dissociationpeak of the enzyme inhibitor (see, e.g., FIGS. 3-6).

In general, the DNA polymerase inhibitors of the present teachings maybe used in any amplification method in which a DNA polymerase isemployed. For example, the disclosed DNA polymerase inhibitors can beused in one or more of the following methods: DNA sequencing, DNAamplification, RNA amplification, reverse transcription, DNA synthesisand/or primer extension. The disclosed DNA polymerase inhibitors can beused in reaction compositions for amplifying target nucleic acids byprimer extension, for example but not limited to, PCR and/or reversetranscription. The DNA polymerase inhibitors of the current teachingscan also be used in certain sequencing techniques. The disclosed DNApolymerase inhibitors can be used in tests for single nucleotidepolymorphisms (SNPs) by single nucleotide primer extension usingterminator nucleotides. Any such procedures including variationsthereof, for example but not limited to, polynucleotide or primerlabeling, mini-sequencing and the like are contemplated for use with theDNA polymerase inhibitors disclosed herein.

In some embodiments, a ligase inhibitor comprises an oligonucleotidethat can serve, at a first temperature, as a ligation substrate mimic,that is a substrate comprising a nick that can not be ligated by theligase. In some embodiments, a ligase inhibitor comprises two adjacentlyhybridized nucleic acid ends, but at least one terminal nucleotide of atleast one of the ends is not hybridized to the “template” strand of theinhibitor and the two ends can not be ligated together. In certainembodiments, a ligase inhibitor comprises two adjacently hybridizednucleic acid ends, but at least one end comprises a terminal nucleotidethat is not ligatable by the ligase. For example, the 3′ terminalnucleotide does not comprise a 3′-hydroxyl group, the 5′ terminalnucleotide does not comprise a 5′-phosphate group. or both. Anillustrative ligase inhibitor embodiment comprising a nick that can notbe closed by a ligase is shown in FIG. 1 E. This exemplary ligaseinhibitor comprises a first region (1), a second region (2), a thirdregion (3), a fourth region (4), a fifth region (8), and a sixth region(9). The second region (2), shown as a loop structure, further comprisesa first quencher (6); and the fifth region (8), also shown as a loopstructure, further comprises a second quencher (7). The first region (1)is shown annealed with the third region (3) to form a firstdouble-stranded segment; and the fourth region (4) is shown annealedwith the sixth region (9) to form a second double-stranded segment, forexample, as can occur at the first temperature. The 3′-end of the sixthregion (9) comprises a non-ligatable end (10), for example but notlimited to, a terminal nucleotide that lacks a 3′—OH group (shown as X).In certain embodiments, either the 3′-end of the sixth region and/or the5′-end of the first region of such an illustrative ligase inhibitor isnot annealed with the “template strand” (in this illustration, thefourth region (4) and/or the third region (3), respectively. In certainembodiments, the upstream end of a ligase inhibitor (shown as 9 in theillustrative ligase inhibitor depicted in FIG. 1E) comprises 8nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, or more than 20 nucleotides. It is to be appreciated thatthe length of the “upstream strand” of a ligase inhibitor will typicallybe designed to be at least as long as the footprint of the desiredligase and may be longer,

In certain embodiments, a ligase inhibitor comprises twooligonucleotides that can adjacently hybridize with a template strand,but the opposed ends at the nick are not suitable for ligation together,for example but not limited to the 3′-end of the upstream strand doesnot comprise a 3′—OH group, the 5′-end of the downstream strand does notcomprise a 5′-phosphate group, or both.

Some ligase inhibitor embodiments comprise at least threeoligonucleotides, a first oligonucleotide, a second oligonucleotide, anda third oligonucleotide, wherein the first oligonucleotide comprises afirst region, the second oligonucleotide comprises a third region and afourth region, and the third oligonucleotide comprises a sixth region,wherein the first region is complementary with the third region and thefourth region is complementary with the sixth region.

Certain ligase inhibitors comprise two oligonucleotides, including afirst oligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a first region, a second region, a thirdregion and a fourth region and the second oligonucleotide comprises asixth region, and wherein the first region is complementary with thethird region and the fourth region is complementary with the sixthregion. Under appropriate conditions, including at a first temperature,the first region and the third region can anneal and form at least onedouble-stranded segment and the fourth region and the sixth region cananneal to form at least one double-stranded segment. Other ligaseinhibitor embodiments comprise more than two oligonucleotides that can,under appropriate conditions including at a first temperature, anneal toform hybridization structure comprising a nick or a gap between twoadjacently hybridized oligonucleotide ends that can not be closed by aligase. In certain ligase inhibitor embodiments, at least one nucleotideat or near the 3-end of the upstream oligonucleotide, the 5′-end of thedownstream oligonucleotide, or both, is not complementary with thecorresponding nucleotide(s) of the third oligonucleotide, to which thefirst and second oligonucleotides adjacently anneal. Thus at least oneof the opposing ends is not efficiently annealed with the template andthe ligase is unable to ligate them together.

In certain embodiments, a cleaving enzyme inhibitor comprises a flapsequence comprising at least one internucleotide linkage that is notcleavable or is slowly cleaved by the cleaving enzyme. An exemplaryembodiment of such an inhibitor is shown schematically in FIG. 1 F. Theillustrative inhibitor comprises a first region (1), a second region(2), a third region (3) comprising a first quencher (6), a fourth region(4), a fifth region (8), and a sixth region (9) that in thisillustrative inhibitor comprises a second quencher (7). The first region(1) and the third region (3) are shown annealed to form a firstdouble-stranded segment, the fourth region (4) and the sixth region (9)are annealed to form a second double-stranded segment, and the secondregion (2) and the fifth region (8) are each shown as loop structures.Upstream from the first region (1) is a flap sequence (11) that in thisexemplary embodiment, comprises a multiplicity of internucleotidelinkages that can not be cleaved by the cleaving enzyme (12). In thisconformation, the illustrative enzyme inhibitor forms a cleavagestructure mimic, that is a secondary structure that resembles a nucleicacid cleavage structure but which serves as an ineffective substrate forthe cleaving enzyme. In certain embodiments, for example but not limitedto, when the cleaving enzyme comprises a DNA polymerase withpolymerization activity and/or when the reaction composition comprises acleaving enzyme and a DNA polymrase, the 3′-end of the cleaving enzymeinhibitor comprises a non-extendible nucleotide, including withoutlimitation a ddN.

In certain embodiments, the nucleotide sequence of an enzyme inhibitorcomprises an aptamer that comprises at least one double-stranded segmentand that binds to and inhibits the enzymatic activity of the enzyme whenbound by the aptamer. When the aptamer is free in solution below thesecond temperature or is bound to the enzyme in a complex, the quencherabsorbs at least some of the fluorescent signal generated by nucleicacid dye molecules associated with the aptamer.

Certain Exemplary Complexes

A complex according to the present teachings comprises an enzymeinhibitor associated with an enzyme such that at least one enzymaticactivity of the enzyme is inhibited. In certain embodiments, a complexcomprises an enzyme inhibitor associated with an amplifying enzyme, forexample, any enzyme that is included in an amplification reaction. Insome embodiments, a complex comprises an RNA polymerase associated withan RNA polymerase inhibitor. In some embodiments, a complex comprises aligase inhibitor associated with a ligase. In some embodiments, acomplex comprises a helicase inhibitor associated with a helicase. Incertain embodiments, a complex comprises a cleaving enzyme associatedwith a cleaving enzyme inhibitor. Some complexes further compriseadditional components, for example but not limited to, adeoxyribonucleotide (dNTP), a ribonucleotide (rNTP), a nucleotideanalog, a helicase accessory protein, an SSB, or an enzyme cofactorincluding without limitation, ATP and nicotinamide adenine dinucleotide(NAD+), and including non-cleavable analogs thereof that can participatein the formation and/or stabilization of certain enzyme-enzyme inhibitorcomplexes, or combinations thereof.

In certain embodiments, an enzyme-enzyme inhibitor complex comprises aDNA polymerase associated with a DNA polymerase inhibitor. In certainembodiments, a complex comprising a DNA polymerase inhibitor and a DNApolymerase further comprises a NTP and/or a nucleotide analog that canparticipate in the DNA polymerase inhibitor-DNA polymerase complex.According to the present teachings, when a DNA polymerase is complexed(i.e., associated in a complex) with a DNA polymerase inhibitor andoptionally a NTP and/or a nucleotide analog, the enzymatic activity ofthe DNA polymerase with respect to its ability to catalyze the additionof nucleotides to the 3′-end of a primer or a nascent polynucleotidestrand is inhibited. Typically, the disclosed DNA polymerase inhibitorsare designed to form at least one double-stranded segment and complexwith a DNA polymerase at a first temperature. When the complex is heatedto a second temperature, the double-stranded segment of the DNApolymerase inhibitor denatures and the complex dissociates. Whenreleased from the complex, the synthetic activity of the DNA polymeraseis restored and, under appropriate conditions, certain nucleic acidsequences can be amplified.

According to certain embodiments, a complex comprises a DNA polymeraseinhibitor associated with a DNA polymerase such that the enzymaticactivity of the DNA polymerase is inhibited. In some embodiments, acomplex comprises a DNA polymerase inhibitor in a single or doublestem-loop conformation associated with a DNA polymerase. In someembodiments, a complex comprises a DNA polymerase associated with a DNApolymerase inhibitor comprising at least two oligonucleotides that areannealed to form at least one double-stranded segment.

Typically, the first and third regions of a DNA polymerase inhibitoranneal to form a double-stranded segment at the first temperature andthe DNA polymerase inhibitor assumes a stem-loop conformation or aduplex conformation, as appropriate. In certain embodiments, the fourthand sixth regions of a DNA polymerase inhibitor anneal to form adouble-stranded segment at the first temperature and the DNA polymeraseinhibitor assumes a stem-loop conformation, a double stem-loopconformation, or a duplex conformation, as appropriate. When a DNApolymerase inhibitor in a stem-loop or a duplex conformation is combinedwith a DNA polymerase, the DNA polymerase inhibitor and the DNApolymerase can associate to form a complex, wherein the DNA polymeraseactivity is inhibited. As the reaction temperature is increased, thedouble-stranded segment(s) of the DNA polymerase inhibitors denature ator near the second temperature, causing the complex to dissociate andreleasing the inhibition of the DNA polymerase.

The DNA polymerases of the current teachings typically include but arenot limited to, DNA-dependent DNA polymerases and RNA-dependent DNApolymerases, including reverse transcriptases. Certain reversetranscriptases possess DNA-dependent DNA polymerase activity undercertain reaction conditions, including AMV reverse transcriptase andMMLV reverse transcriptase. Such reverse transcriptases withDNA-dependent DNA polymerase activity may be suitable for use with thedisclosed methods and are expressly within the contemplation of thecurrent teachings. Descriptions of DNA polymerases can be found in,among other places, Lehninger Principles of Biochemistry, 3d ed., Nelsonand Cox, Worth Publishing, New York, N.Y., 2000, particularly Chapters26 and 29; Twyman, Advanced Molecular Biology: A Concise Reference, BiosScientific Publishers, New York, N.Y., 1999; Ausubel et al.; Lin andJaysena, J. Mol. Biol. 271:100-11, 1997; Pavlov et al., Trends inBiotechnol. 22:253-60, 2004; and Enzymatic Resource Guide: DNApolymerases, 1998, Promega, Madison, Wis.

Inhibition of DNA polymerase activity can be observed with respect tothe synthesis of secondary amplicons or more generally, with respect tooverall nucleic acid synthesis by the DNA polymerase. In general, one ofskill in the art may choose to optimize synthesis of desired ampliconswhile minimizing synthesis of spurious side-products. Hence, whengenerating a desired amplicon, the disclosed DNA polymerase inhibitorscan inhibit synthesis of secondary amplicons by about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or greater than about 99%, when compared to the amount of secondaryamplicons synthesized in the absence of the selected DNA polymeraseinhibitor.

Inhibition of ligase activity can be observed with respect to thesynthesis of undesired side-products, including without limitation,misligation products, or more generally, with respect to overall nucleicacid amplification in the reaction composition, for example but notlimited to, a reaction composition in which LCR, LDR, LDR-PCR, PCR-LDR,or FEN-LCR occurs. In general, one of skill in the art may choose tooptimize synthesis of desired amplicons while minimizing synthesis ofspurious side-products. Hence, when generating a desired amplicon, thedisclosed ligase inhibitors can inhibit synthesis of undesired sideproducts by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about96%, about 97%, about 98%, about 99%, or greater than about 99%, whencompared to the amount of secondary amplicons synthesized in the absenceof the selected ligase inhibitor.

Inhibition of cleaving enzyme activity can be observed with respect tothe synthesis of undesired side-products or more generally, with respectto overall nucleic acid amplification in the reaction composition, forexample but not limited to a reaction composition in which FEN-LCRoccurs. In general, one of skill in the art may choose to optimizesynthesis of desired amplicons while minimizing synthesis of spuriousside-products. Hence, when generating a desired amplicon, the disclosedcleaving enzyme inhibitors can inhibit synthesis of undesired sideproducts by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about96%, about 97%, about 98%, about 99%, or greater than about 99%, whencompared to the amount of secondary amplicons synthesized in the absenceof the selected cleaving enzyme inhibitor.

Inhibition of helicase activity can be observed with respect to thesynthesis of secondary amplicons or more generally, with respect tooverall nucleic acid synthesis in the reaction composition, for examplebut not limited to a reaction composition in which HDA occurs. Ingeneral, one of skill in the art may choose to optimize synthesis ofdesired target nucleic acids while minimizing synthesis of spuriousside-products. Hence, when generating a desired amplicon, the disclosedhelicase inhibitors can inhibit synthesis of secondary amplicons byabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 85%, about 90%, about 95%, about 96%, about97%, about 98%, about 99%, or greater than about 99%, when compared tothe amount of secondary amplicons synthesized in the absence of theselected helicase inhibitor.

The disclosed enzyme inhibitors can be combined with the enzymes in avariety of ratios or concentrations to form complexes. In someembodiments, the enzyme inhibitor is present at a larger molarconcentration than the enzyme. In other embodiments, the enzymeinhibitor is present at about the same or a lesser molar concentrationthan the enzyme. One of skill in the art may choose to use a molar ratioof enzyme inhibitor to enzyme that is greater than 1:1 (inhibitor:enzyme) in order to insure that sufficient enzyme inhibitor is presentso that every enzyme molecule can associate with an enzyme inhibitor toform a complex. In general, highly effective enzyme inhibitors may beused at lower concentrations than less effective enzyme inhibitors.Hence, enzyme inhibitors can be provided in a reaction composition at avariety of concentrations. Such concentrations can vary, for example,from about 1 nM to about 10 mM, or from about 5 nM to about 1 mM, orfrom about 10 nM to about 100 μM, or other convenient concentrationsselected by one of skill in the art.

The enzyme inhibitors disclosed herein can be combined with an enzymeeither before or during an amplification reaction to form a complexprovided that the amplification reaction conditions comprise at leastone step at the first temperature. In certain embodiments, an enzyme andan enzyme inhibitor are combined at the first temperature before thereaction composition is formed. Such a pre-incubation step mayfacilitate the formation of an enzyme inhibitor-enzyme complex and helpdecrease or eliminate the synthesis of undesired side-products such asmis-primed amplicons, misligated probes, and oligomerized primers.

Certain Exemplary Methods

The disclosed enzyme inhibitors serve at least two functions in themethods of the current teachings. First, the disclosed enzyme inhibitorsserve to inhibit the enzymatic activity of a corresponding enzyme at afirst temperature, decreasing secondary amplicon formation due to, amongother things, mis-annealing or primers and/or probes to sequences otherthan target nucleic acids and primer dimer formation. Those in the artwill appreciate that by decreasing the formation of secondaryamplification products, the enzyme inhibitors of the current teachingscan reduce the non-specific fluorescence of the reaction composition.Second, the disclosed enzyme inhibitors can also reduce the non-specificfluorescence in the reaction composition due to the self-quenchingability of the enzyme inhibitor, as the at least one quencher moiety canabsorb at least some of the fluorescence emitted by the nucleic acid dyemolecules associated with the double-stranded segment(s) of the enzymeinhibitor. In certain embodiments, enzyme inhibitors also increase theamplicon yield in the disclosed methods.

Methods for amplifying a target nucleic acid are provided. According tocertain method embodiments, a reaction composition is formed at a firsttemperature, wherein the reaction composition comprises a DNApolymerase, a DNA polymerase inhibitor comprising a nucleotide sequenceand a quencher, a nucleoside triphosphate (NTP), typically a mixture ofdeoxyribonucleotide triphosphates (dNTPs), a target nucleic acid, aprimer, and a nucleic acid dye. In certain embodiments, a reactioncomposition further comprises a nucleotide analog. In some embodiments,the DNA polymerase, the DNA polymerase inhibitor, and optionally a NTPand/or a nucleotide analog, are combined prior to forming the reactioncomposition. In certain embodiments, the DNA polymerase and the DNApolymerase inhibitor are pre-incubated at the first temperature prior toforming the reaction composition. At the first temperature, thenucleotide sequence of the DNA polymerase inhibitor comprises at leastone double-stranded segment and the DNA polymerase and the DNApolymerase inhibitor can associate to form a complex. The quencherabsorbs at least some of the fluorescent signal emitted by the nucleicacid dye molecules associated with the double-stranded segment of thenucleotide sequence, relative to the signal that is detected in aparallel reaction composition comprising the same DNA polymeraseinhibitor nucleotide sequence but lacking the quencher(s). The reactioncomposition is then heated to a second temperature that is near, at, orabove the melting temperature of the DNA polymerase inhibitor, causingthe double-stranded segment to denature and dissociating the complex.With the release of the DNA polymerase inhibitor, from the complex, theenzymatic activity of the DNA polymerase is no longer inhibited. Thereaction composition is subjected to at least one cycle of amplificationto generate a multiplicity of amplicons.

Methods for reducing non-specific fluorescence in a reaction compositionare provided. According to certain such methods, a reaction compositionis formed at a first temperature, wherein the reaction compositioncomprises a DNA polymerase, a DNA polymerase inhibitor comprising anucleotide sequence and a quencher, a NTP, typically a mixture of dNTPs,a target nucleic acid, a primer, and a nucleic acid dye. In certainembodiments, a reaction composition further comprises a nucleotideanalog. In some embodiments, the DNA polymerase, the DNA polymeraseinhibitor, and optionally a NTP and/or nucleotide analog, are combinedprior to forming the reaction composition. In certain embodiments, theDNA polymerase and the DNA polymerase inhibitor are pre-incubated at thefirst temperature to form a complex prior to forming the reactioncomposition. The quencher absorbs at least some of the fluorescentsignal emitted by the nucleic acid dye molecules associated with thedouble-stranded segment of the nucleotide sequence, relative to thesignal that is detected in a parallel reaction composition comprisingthe same DNA polymerase inhibitor nucleotide sequence but lacking thequencher(s). The reaction composition is then heated to a secondtemperature that is near, at, or above the melting temperature of theDNA polymerase inhibitor, causing the double-stranded segment todenature and dissociating the complex. With the release of the DNApolymerase, from the complex, the polymerization activity of the DNApolymerase is no longer inhibited. The reaction composition is subjectedto at least one cycle of amplification to generate a multiplicity ofamplicons. Under appropriate detection conditions, the fluorescence ofthe nucleic acid dye associated with the multiplicity of amplicons inthe reaction composition can be detected, while the fluorescence of thenucleic acid dye associated with the double-stranded segment of thenucleotide sequence of the DNA polymerase inhibitor is at least reducedby the quencher.

In some embodiments, the at least one cycle of amplification comprises amultiplicity of cycles of amplification, for example but not limited to,at least 10 cycles, at least 15, cycles, at least 20 cycles, at least 25cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, ormore than 40 cycles of amplification. In some embodiments, thesubjecting the reaction composition to at least one cycle ofamplification comprises PCR, including variations of PCR, for examplebut not limited to, RT-PCR, asymmetric PCR, or quantitative or real-timePCR (see, e.g., Rapley, particularly Part VII; Protocols & ApplicationsGuide, rev. 9/04, Promega; McPherson).

Certain embodiments of the disclosed methods comprise a multiplexamplification step, including but not limited to a multiplicity ofparallel single-plex or lower plexy amplification reactions (for example2-plex, 3-plex, 4-plex, 5-plex, or 6-plex amplification reactions), amultiplex detection step, including but not limited to a multiplicity ofparallel single-plex of lower plexy detection steps (for example whereintwo, three, four, five, or six different amplicons are detected in thesame reaction composition), or both a multiplex amplification reactionand a multiplex detection procedure. In some embodiments, the targetnucleic acid comprises a multiplicity of different target nucleic acids,the primer comprises a multiplicity of different primers or amultiplicity of different primer pairs, the multiplicity of ampliconscomprises a multiplicity of different amplicons, and the detectingcomprises detecting the fluorescence of the nucleic acid dye associatedwith the multiplicity of different amplicons.

The degree of enzymatic inhibition obtained using the disclosed DNApolymerase inhibitors can vary and may depend upon the method employed,the DNA polymerase, the structure and melting point of the selected DNApolymerase inhibitor and other factors such as the primer extensiontemperature. Each of these variables can be optimized by one of skill inthe art to using the teachings herein and/or available procedures toobtain optimal production of the desired product with minimal productionor non-target nucleic acids. Likewise, the level of non-specificfluorescence reduction can vary, depending upon, among other things, theparticular quencher(s) in the nucleotide sequence, the number ofquenchers employed per DNA polymerase inhibitor, the nucleic acid dyeemployed, the reaction conditions, and the effectiveness of the DNApolymerase inhibitor at decreasing the amount on secondary amplificationproducts. Those in the art will appreciate that the number and placementof a particular quencher or quenchers in a particular DNA polymeraseinhibitor, the pairing of a particular DNA polymerase with a particularDNA polymerase inhibitor, and the pairing of a particular quencher witha particular nucleic acid dye, can be evaluated empirically usingroutine methods known in the art and without undue experimentation tooptimize the reduction of non-specific fluorescence in a particularreaction composition and amplification technique.

According to certain method embodiments, a ligase forms a complex with aligase inhibitor at a first temperature in a reaction compositioncomprising a target nucleic acid and a ligation probe pair. In certainembodiments, the ligase and the ligase inhibitor are combined andpre-incubated prior to forming a reaction composition. At a first secondtemperature, the ligase-ligase inhibitor complex dissociates, releasingthe ligase. The upstream and downstream ligation probes of the ligationprobe pair selectively hybridize with the target nucleic acid and theligase catalyzes the formation of a ligated probe. Some such embodimentscomprise a multiplicity of cycles of amplification comprising the stepsof denaturing, annealing the upstream and downstream ligation probes,and ligating the probes to generate a ligated probe. In certainembodiments, the reaction composition comprises a ligation probe pairthat is designed to specifically hybridize with at least a portion ofthe complement of a ligated probe. In some embodiments, a ligated probecomprises a primer-binding site and the reaction composition comprises aprimer and a DNA polymerase-DNA polymerase complex.

According to certain disclosed methods, a cleaving enzyme forms acomplex with a cleaving enzyme inhibitor and a ligase forms a complexwith a ligase inhibitor at a first temperature. In certain embodiments,at a first second temperature, the cleaving enzyme-cleaving enzymeinhibitor complex dissociates. The released cleaving enzyme can thencleave flap portions from certain overlap flap structures comprising (1)a target nucleic acid or a single-stranded amplicon, (2) a upstreamcleavage probe, and (3) a corresponding downstream cleavage probe thatcomprises a 5′-overhang or flap sequence that overlaps the 3′-end of theupstream cleavage probe by at least one nucleotide. When the flap iscleaved by the cleaving enzyme, a hybridization structure comprising thetemplate strand, the upstream cleavage probe, and the hybridizedfragment of the downstream cleavage probe, with a ligatable nick betweenthe 3′-end of the upstream cleavage probe and the 5′-end of thehybridized fragment of the downstream cleavage probe. In someembodiments, at a second second temperature the ligase-ligase inhibitorcomplex dissociates and the released ligase can ligate the nick in thehybridization structure to generate a duplex comprising a ligated probeand a template strand. In certain embodiments, a ligated probe comprisesat least one primer-binding site. Those in the art will appreciate thatthe first second temperature and the second second temperature can beapproximately the same temperatures or they can be differenttemperatures.

Some method embodiments further comprise a DNA polymerase-DNA polymeraseinhibitor complex at a first temperature. At an appropriate third secondtemperature the DNA polymerase-DNA polymerase inhibitor complexdissociates. Under suitable conditions, a primer specifically hybridizeswith the primer-binding portion of a ligated probe and primer extensioncan occur. Those in the art will appreciate that when different enzymeinhibitors are employed in a reaction composition, at least two of: thefirst second temperature, the second second temperature, and the thirdsecond temperature can be approximately the same temperatures or theycan all be different temperatures.

Exemplary cleaving enzymes for use in the disclosed complexes, methodsand kits include without limitation, E. coli DNA polymerase I, Thermusaquaticus DNA polymerase I, Thermus thermophilus DNA polymerase I,mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcus jannaschiiFEN-1, Pyrococcus furiosus FEN-1, Methanobacterium thermoautotrophicumFEN-1, Thermus thermophilus FEN-1, Cleavase® enzymes (Third Wave, Inc.,Madison, Wis.), Saccharomyces cerevisiae RTH1, S. cerevisiae RAD27Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′ exonuclease,Pyroccus horikoshii FEN-1, human exonuclease 1, calf thymus 5′-3′exonuclease, including homologs thereof in eubacteria, eukaryotes, andarchaea, such as members of the class II family of structure-specificenzymes. Descriptions of cleaving enzymes can be found in, among otherplaces, Lyamichev et al., Science 260:778-83 (1993); Eis et al., Nat.Biotechnol. 19:673-76 (2001); Shen et al., Trends in Bio. Sci. 23:171-73(1998); Kaiser et al. J. Biol. Chem. 274:21387-94 (1999); Ma et al., J.Biol. Chem. 275:24693-700 (2000); Allawi et al., J. Mol. Biol.328:537-54 (2003); Sharma et al., J. Biol. Chem. 278:23487-96 (2003);and Feng et al., Nat. Struct. Mol. Biol. 11:450-56 (2004).

According to certain disclosed methods, a DNA polymerase is combinedwith a DNA polymerase inhibitor, and optionally a NTP and/or anucleotide analog, to form a complex. In certain embodiments, the DNApolymerase comprises a reverse transcriptase, a DNA-dependent DNApolymerase, including without limitation a thermostable DNA polymerase,or a reverse transcriptase and a DNA-dependent DNA polymerase. In someembodiments, the DNA polymerase inhibitor comprises (1) a first DNApolymerase inhibitor that can form a complex with the reversetranscriptase at a suitable first temperature, (2) a second DNApolymerase inhibitor that can form a complex with the DNA-dependent DNApolymerase at a suitable first temperature, or (3) a first DNApolymerase inhibitor that can form a complex with the reversetranscriptase at a suitable first temperature and a second DNApolymerase inhibitor that can form a complex with the DNA-dependent DNApolymerase at a suitable first temperature, wherein the first DNApolymerase inhibitor and the second DNA polymerase inhibitor comprisethe same nucleotide sequence or a different nucleotide sequence, andwherein the suitable first temperature for the first DNA polymeraseinhibitor and the suitable first temperature for the second DNApolymerase inhibitor are the same temperature or different temperatures.

According to certain disclosed methods, amplification comprises a twophase PCR reaction comprising two different reaction compositions, afirst reaction composition and a second reaction composition, eachcomprising a DNA polymerase and a DNA polymerase inhibitor. In certainsuch embodiments, a first reaction composition comprises a first DNApolymerase, a first DNA polymerase inhibitor, a NTP, typically a mixtureof NTPs, and a primer, typically a multiplicity of different primerpairs. In certain embodiments, the DNA polymerase, the DNA polymeraseinhibitor, and optionally a NTP and/or a nucleotide analog are combinedprior to forming the first reaction composition. The first reactioncomposition is subjected to a limited number of cycles of amplification,for example but not limited to two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen cyclesof amplification. The first reaction composition is diluted after thelimited first stage amplification and a portion of the diluted firstreaction composition is combined with a second DNA polymerase, a secondDNA polymerase inhibitor, a NTP, typically a mixture of NTPs, and aprimer, typically a primer pair. In certain embodiments, the DNApolymerase, the DNA polymerase inhibitor, and optionally a NTP and/or anucleotide analog, are combined prior to forming the second reactioncomposition. The second reaction composition is subjected to amultiplicity of cycles of amplification, for example but not limited to,10-45 cycles of amplification or 20-40 cycles of amplification,including any number of cycles of amplification in the listed ranges, asif each and every number of cycles was expressly recited herein. In someembodiments, there is enough residual first DNA polymerase in thediluted first reaction composition that a second DNA polymerase is notnecessary. In some embodiments, there is enough residual first DNApolymerase inhibitor in the diluted first reaction composition that asecond DNA polymerase inhibitor is not necessary. In certainembodiments, the first DNA polymerase and the second DNA polymerase arethe same polymerase or different polymerases, including withoutlimitation, a reverse transcriptase and a DNA-dependent DNA polymerase.In some embodiments, the first DNA polymerase inhibitor and the secondDNA polymerase inhibitor are the same inhibitor or a differentinhibitor. For illustration purposes but not as a limitation of suchembodiments, consider an exemplary RT-PCR reaction that comprises afirst reaction comprising a reverse transcriptase, a first DNApolymerase inhibitor, and optionally, a NTP and/or a nucleotide analogand a second reaction composition comprising a thermostableDNA-dependent DNA polymerase, a second DNA polymerase inhibitor, andoptionally a NTP and/or a nucleotide analog. The first DNA polymeraseinhibitor can be designed to inhibit the reverse transcriptase activityat temperatures below the optimal temperature for reverse transcription(i.e., an exemplary first phase first temperature), but not at or abovethe optimal reverse transcription temperature (i.e., an exemplary firstphase second temperature). The second DNA polymerase inhibitor can bedesigned to inhibit the enzymatic activity of the thermostable DNApolymerase at temperatures below the second phase first temperature, forexample but not limited to, a temperature about 5° C. to about 10° C.below or about 4° C. below to about 6° C. below the Tm of at least oneof the PCR primers (i.e., an exemplary second phase first temperature),but not above the Tm of the PCR primers (i.e., an exemplary first phasesecond temperature).

The methods of the current teachings can typically be used with anytarget nucleic acid. The disclosed methods are useful not only forproducing large amounts of a desired amplicon, but also for producing orsequencing nucleic acids that are known to exist but are not completelysequenced or purified. One need know only the identity of a sufficientnumber of bases at one or two ends of the target, i.e., a targetflanking sequence, in sufficient detail so that at least one primer canbe prepared that can serve as a sequencing primer. After sequencing andidentification of an acceptable second target flanking sequence, asecond primer can be made and the target nucleic acid lying between theflanking sequences can be exponentially amplified and in someembodiments, quantified. In other embodiments, when sufficient sequencehas been obtained, an appropriate ligation probe set and/or anappropriate cleavage probe set can be synthesized.

In certain embodiments of the disclosed methods, detecting comprisesevaluating an internal standard or a control sequence, and may includecomparing the quantity of a desired amplicon with a standard curve or aninternal size standard. In some embodiments, a control sequence, apassive reference dye, or both are included in a reaction composition toaccount for lane-to-lane, capillary-to-capillary, and/or assay-to-assayvariability.

Certain embodiments of the current methods further comprise a multi-wellreaction vessel, including without limitation, a multi-well plate or amulti-chambered microfluidic device, in which a multiplicity ofamplification reactions and, in some embodiments, detection areperformed, typically in parallel. In certain embodiments, one or moremultiplex reactions for generating amplicons are performed in the samereaction vessel, including without limitation, a multi-well plate, suchas a 96-well, a 384-well, a 1536-well plate, and so forth; or amicrofluidic device, for example but not limited to, a TaqMan® LowDensity Array (Applied Biosystems). In some embodiments, a massivelyparallel amplifying step comprises a multi-well reaction vessel,including a plate comprising multiple reaction wells, for example butnot limited to, a 24-well plate, a 96-well plate, a 384-well plate, or a1536-well plate; or a multi-chamber microfluidics device, for examplebut not limited to a TaqMan Low Density Array wherein each chamber orwell comprises an appropriate primer(s), primer set(s), and/or reporterprobe(s), as appropriate. Typically such amplification steps occur in aseries of parallel single-plex, two-plex, three-plex, four-plex,five-plex, or six-plex reactions, although higher levels of parallelmultiplexing are also within the intended scope of the currentteachings.

In certain embodiments, the reaction composition further comprises apassive reference dye. The passive reference dye is included in thereaction composition as an internal control to allow for normalizationof non-PCR related variations in fluorescence, for example but notlimited to, well-to-well, tube-to-tube, plate-to-plate, andassay-to-assay variation. The passive reference provides a baseline fornormalization because its fluorescence does not change during the courseof the amplification reaction. Typically, the passive reference does notinterfere with amplification reactions. The use of a passive referencedye and normalization calculations based on the passive reference, forexample but not limited to, Rn and ΔRn, are well known in the art (see,e.g., Killigore et al., J. Clin. Micro., 38:2516-19, 2000; TaqMan® PCRReagent Kit With AmpliTaq Gold® DNA polymerase Protocol, AppliedBiosystems P/N 402823 Rev. D 2003; Brilliant® SYBR® Green QRT-PCR MasterMix Kit, 1-step Instruction Manual, Rev. #75003a, Stratagene, 2005; andEssential of Real Time PCR, Applied Biosystems). In some embodiments,the passive reference dye comprises ROX™ or TAMRA™.

Certain Exemplary Kits

The instant teachings also provide kits designed to expedite performingcertain of the disclosed methods. Kits may serve to expedite theperformance of certain disclosed methods by assembling two or morecomponents required for carrying out the methods. In certainembodiments, kits contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits include instructions for performing one or more of the disclosedmethods. Preferably, the kit components are optimized to operate inconjunction with one another.

Certain disclosed kits comprise an enzyme inhibitor comprising anucleotide sequence and a quencher. In certain embodiments, kitscomprise at least one of: a ligase inhibitor, a helicase inhibitor, aRNA polymerase inhibitor, a cleaving enzyme inhibitor, and/or a DNApolymerase inhibitor. Certain kits of the current teachings furthercomprise at least one of: a ligase, a helicase, an RNA polymerase, and acleaving enzyme. Certain kits comprise an enzyme inhibitor and furthercomprise at least one of: a primer, including without limitation arandom primer or a primer comprising oligo dT, or a primer pair; aligation probe pair; a cleavage probe set; a ligase cofactor includingwithout limitation, ATP or NAD; an SSB; and/or a helicase accessoryprotein. In some embodiments, kits comprise a primer, a DNA polymerase,a ligase, or combinations thereof. In certain embodiments, kits comprisea NTP, a nucleotide analog, or both.

Certain kit embodiments comprise a DNA polymerase inhibitor comprising anucleotide sequence and a quencher. In certain embodiments, a kitcomprises a DNA polymerase; a control sequence, for example but notlimited to an internal standard sequence such as a housekeeping geneand/or a coamplification sequence (see, e.g., Siebert and Larrick,BioTechniques 14:244-49 (1993); Joyce, Quantitative RT-PCR, 83-92, inMethods in Mol Biol., vol. 193, O'Connell, ed., Humana Press;Raeymaekers, Mol. Biotechnol. 115-22 (2000)) or a polynucleotide laddercomprising molecular size or weight standards; a primer and/or a primerpair; a reporter probe; a nucleic acid dye; a passive reference dye; orcombinations thereof. In certain embodiments, kits comprise amultiplicity of different primer pairs. In some embodiments, kitscomprise a forward primer, a reverse primer, or a forward primer and areverse primer, that further comprises a reporter group. In some suchembodiments, the reporter group of a forward primer of a primer pair isdifferent from the reporter group of the reverse primer of the primerpair.

The skilled artisan will appreciate that many different species ofreporter groups can be used in the present teachings, eitherindividually or in combination with one or more different reportergroup. In certain embodiments, a reporter group emits a fluorescent, achemiluminescent, a bioluminescent, a phosphorescent, or anelectrochemiluminescent signal. Some non-limiting examples of reportergroups include fluorophores, radioisotopes, chromogens, enzymes,antigens including but not limited to epitope tags, semiconductornanocrystals such as quantum dots, heavy metals, dyes, phosphorescencegroups, chemiluminescent groups, electrochemical detection moieties,binding proteins, phosphors, rare earth chelates, transition metalchelates, near-infrared dyes, electrochemiluminescence labels, and massspectrometer-compatible reporter groups, such as mass tags, charge tags,and isotopes (see, e.g., Haff and Smirnov, Nucl. Acids Res. 25:3749-50,1997; Xu et al., Anal. Chem. 69:3595-3602, 1997; Sauer et al., Nucl.Acids Res. 31:e63, 2003). Detailed protocols for attaching reportergroups to nucleic acids can be found in, among other places, Hermanson,Bioconjugate Techniques, Academic Press, San Diego, 1996; CurrentProtocols in Nucleic Acid Chemistry, Beaucage et al., eds., John Wiley &Sons, New York, N.Y. (2000), including supplements through August 2005;and Haugland, Handbook of Fluorescent Probes and Research Products,10^(th) ed., Molecular Probes-Invitrogen, 2005.

In certain embodiments, a kit comprises two or more different enzymeinhibitors, for example but not limited to a ligase inhibitor and acleaving enzyme inhibitor; a cleaving enzyme inhibitor, a ligaseinhibitor, and a DNA polymerase inhibitor; or a helicase inhibitor and aDNA polymerase inhibitor. In some embodiments, a kit comprises two ormore different DNA polymerase inhibitors. In certain embodiments, kitscomprise two different enzymes, including without limitation, aDNA-dependent DNA polymerase and an RNA-dependent DNA polymerase, suchas a reverse transcriptase; a ligase and a cleaving enzyme; an RNApolymerase and a DNA polymerase, for example but not limited to, areverse transcriptase; and a helicase and a DNA polymerase. In certainembodiments, a kit comprises a thermostable DNA polymerase.

The current teachings, having been described above, may be betterunderstood by reference to examples. The following examples are intendedfor illustration purposes only, and should not be construed as limitingthe scope of the teachings herein in any way.

Example 1

To evaluate the effect of the quencher moiety of certain illustrativeenzyme inhibitors to absorb at least some of the fluorescence emittedfrom nucleic acid dye molecules associated with double-stranded segmentsof the illustrative enzyme inhibitors, five exemplary DNA polymeraseinhibitors were synthesized, as shown in Table 1 (below). The identity,location, and number of quencher moieties were varied.—

TABLE 1 Designation Sequence (shown in 5′ to 3′ orientation) “DNATCTGGGATAATTATGGTAAATATATGTTTTCATATATTTA polymeraseTTATAATTATddC (SEQ ID NO: 1) inhibitor” A DNA(Dabcyl)TCTGGGATAATTATGGTAAATATATGTTTTCA polymeraseTATATTTATTATAATTAT^(dd)C (SEQ ID NO: 1) inhibitor  B DNA(ROX)TCTGGGATAATTATGGTAAATATATGTTTTCATAT polymeraseATTTATTATAATTAT^(dd)C (SEQ ID NO: 1) inhibitor  C DNATCTGGGATAATTATGGTAAATATATGTTTTCATATATTTA polymeraseTTATAATTATC(MGB-NFQ) (SEQ ID NO: 1) inhibitor  D DNATCTGGGATA(deazaA)TT(deazaA)TGGTA(deazaA) polymeraseATATGT(Dabcyl-T)TTC(deazaA)TATTTAT  inhibitor TATAATTATC(MGB-NFQ) (SEQ ID NO: 3) E

A series of parallel compositions, each comprising 1×SYBR Green Inucleic acid dye (Molecular Probes) in 1× reaction buffer (50 mM Trisbuffer, pH 9, 5 mM MgCl₂, 250 μM dATP, dCTP and dGTP, 500 μM dUTP, 60 nMROX passive reference dye, and one of the exemplary DNA polymeraseinhibitors shown in Table 1 at concentrations of 5 nM, 10 nM, 25 nM, 50nM, 75 nM, or 100 nM, as appropriate, were formed at room temperature.As seen in Table 1, “DNA polymerase inhibitor” A, DNA polymeraseinhibitor B, DNA polymerase inhibitor C, and DNA polymerase inhibitor Dshare the same nucleotide sequence except the nucleotide at the 3′-endof DNA polymerase inhibitor D is a C, while the nucleotide at the 3′-endof “DNA polymerase inhibitor” A, DNA polymerase inhibitor B, and DNApolymerase inhibitor C all comprise the nucleotide analogdideoxycytosine (ddC). “DNA polymerase inhibitor” A lacks a quenchermoiety (thus A is not a true DNA polymerase inhibitor of the currentteachings, indicated by the use of quotation marks: “DNA polymeraseinhibitor”); DNA polymerase inhibitor B comprises a DABCYL quenchermoiety at its 5′-end; DNA polymerase inhibitor C comprises a ROXquencher moiety at its 5′-end; and DNA polymerase inhibitor D comprisesa minor groove binder comprising a non-fluorescent quencher (MGB-NFQ) atits 3′-end. DNA polymerase inhibitor E comprises a nucleotide sequencethat includes four deaza-dA nucleotide analogs (shown as deazaA) and twoG:T base pair mismatches in its first and third regions. DNA polymeraseinhibitor E also comprises two quencher moieties, a DABCYL moiety in thesecond region loop and a MGB-NFQ at its 3′-end.

A dissociation curve was generated for each of the compositions using anABI PRISM® 7900HT Real-Time Sequence Detection System instrument(Applied Biosystems) for the temperature range 30° C. to 95° C. Thederivative of fluorescence versus temperature was calculated using theassociated dissociation curve software. As shown in FIG. 3, thedissociation peak obtained from the composition comprising 100 nM “DNApolymerase inhibitor” A (shown as 100 nM A) at the Tm of this nucleotidesequence (approximately 56° C.) was much higher than the dissociationpeaks obtained from the compositions comprising 100 nM, 75 nM, or 50 nMDNA polymerase inhibitor B (shown as 100 nM B, 75 nM B, and 50 nM B,respectively). As demonstrated in FIG. 3, the background fluorescence,presumably attributable to the fluorescent signal emitted from thenucleic acid dye molecules associated with the double-stranded segmentof DNA polymerase inhibitor B, is reduced relative to “DNA polymeraseinhibitor” A.

The dissociation curves obtained from the compositions comprising 100 nM“DNA polymerase inhibitor” A, 100 nM DNA polymerase inhibitor C, 75 nMDNA polymerase inhibitor C, and 50 nM DNA polymerase inhibitor C areshown in FIG. 4. As seen in FIG. 4, the dissociation peak obtained with100 nM “DNA polymerase inhibitor” A is substantially higher that thedissociation peaks associated with 100 nM, 75 nM, or 50 nM of DNApolymerase inhibitor C.

The dissociation curves obtained from the composition comprising 50 nM“DNA polymerase inhibitor” A and the composition comprising 50 nM DNApolymerase inhibitor D are shown in FIG. 5. The dissociation peakobtained from the composition comprising 50 nM “DNA polymeraseinhibitor” A (shown as A in FIG. 5) is substantially higher than thedissociation peak obtained form the composition comprising 50 nM DNApolymerase inhibitor D (shown as D).

FIG. 6 shows the dissociation curves obtained from the compositionscomprising 100 nM, 75 nM, 50 nM, 25 nM, 10 nM or 5 nM “DNA polymeraseinhibitor” A (shown as 100 nM/Std, 75 nM/Std, 50 nM/Std, 25 nM/Std, 10nM/Std, and 5 nM/Std, respectively) and 100 nM, 75 nM, 50 nM, 25 nM, 10nM or 5 nM DNA polymerase inhibitor E. As shown in FIG. 6, thedissociation peaks obtained from each of the compositions comprising“DNA polymerase inhibitor” A are detectably higher and generallysubstantially higher than the dissociation peak(s) obtained from thecomposition comprising DNA polymerase inhibitor E, which are essentiallylost in the “baseline” and not readily distinguishable.

It is to be appreciated that these illustrative DNA polymeraseinhibitors are intended as non-limiting examples of various DNApolymerase inhibitor designs, for example but not limited to, nucleotidesequence variations, with and without a minor groove binder, anddifferent quencher moieties, including without limitation differentnumbers of quenchers per inhibitor, different quencher locations withinthe inhibitor (e.g., 3′-end, 5′-end and internal), and differentspecific quenchers (e.g., DABCYL, ROX, and NFQ). Those in the art willunderstand that various DNA polymerase inhibitor designs are possibleand that a suitable DNA polymerase inhibitor can be obtained by routineevaluation of various designs, informed by the present teachings, foruse with a particular DNA polymerase and a given set of reactionconditions.

Example 2: Inhibition of Secondary Amplicons During PCR Amplification ofan Illustrative Target Nucleic Acid in the Plasminogen ActivatorUrokinase (PAU) Gene of gDNA

To evaluate the inhibitory ability of DNA polymerase inhibitor E in theamplification of a target nucleic acid in gDNA, a PCR reaction wasperformed. Six parallel 20 μL reaction compositions were formed at roomtemperature, with each reaction composition comprising: 40 ng human gDNA(Coriell); a PAU target nucleic acid-specific primer pair comprising2.25 μM forward primer:5′-TGTAAAACGACGGCCAGTTCTCATATTCTCTCATCCTCCTGTCCC-3′(SEQ ID NO: 4) and2.25 μM reverse primer: 5′-CAGGAAACAGCTATGACCAAGCGGCTTTAGGCCCACCT-3′(SEQ ID NO: 5); and a final concentration of either 5, 10, 25, 50, 75 or100 nM DNA polymerase inhibitor E; in 1×PCR buffer (50 mM Tris-HCl, pH9, 250 μM dATP, dCTP and dGTP, 500 μM dUTP, 5 mM MgCl2, 0.6 U AmpliTaqDNA polymerase (Applied Biosystems), 60 nM ROX passive reference dye, 8%glycerol, 0.01% Tween-20, 0.01% NaN3, 1×SYBR Green I nucleic acid dye).A no template control was included in a seventh parallel reactioncomposition comprising the same formulation as the other six, exceptthat there was no gDNA and the final concentration of DNA polymeraseinhibitor E was 50 nM.

The reaction compositions were incubated at room temperature forapproximately 15 min and then thermal cycled in an ABI PRISM® 7900HTReal-Time Sequence Detection System instrument (Applied Biosystems). Thefollowing cycles were used: 95° C. for 2 min, 40 cycles of 96° C. for 5sec and 60° C. for 2 min. To evaluate the amplification productsgenerated in each of the thermocycled reaction compositions, 15 μL ofeach reaction composition was loaded into separate lanes of anon-denaturing 4% agarose E-gel (InVitrogen, Carlsbad, Calif.), alongwith two lanes loaded with a molecular size ladder comprising markers of500 base pairs, 400 base pairs, 300 base pairs, 200 base pairs, and 100base pairs (Low Range DNA Marker, InVitrogen). The reaction compositionswere loaded in lanes of the gel as follows: lane B, 5 nM inhibitor E;lane C, 10 nM inhibitor E; lane D, 25 nM inhibitor E; lane E, 50 nMinhibitor E; lane F, 75 nM inhibitor E; lane G, 100 nM inhibitor E; laneH, 50 nM inhibitor E, no template control. The samples wereelectrophoresed for 15 min, and visualized by ethidium bromide. As shownin FIG. 7, the amount of desired amplicon (11) increased as theconcentration of DNA polymerase inhibitor increased until aconcentration of about 75 nM (lane F). The intensity of the secondaryamplicon bands, by contrast, decreased as the DNA polymerase inhibitorconcentration increased.

Example 3: Inhibition of Secondary Amplicons During PCR Amplification ofan Exemplary Target Nucleic Acid of Human Cytochrome P450 in cDNA

Seven parallel 20 μL reaction compositions were formed at roomtemperature, with each composition comprising: 10 ng universal referencehuman cDNA (Stratagene); a P450 target nucleic acid-specific primer paircomprising 200 nM forward primer: 5′-TGGGAGTCCTGGAAGCAGC-3′ (SEQ ID NO:6) and 200 nM reverse primer: 5′-TGGCTTCTGGTCAACAAGTGC-3′ (SEQ ID NO:7); and a final concentration of either 0, 5, 10, 25, 50, 75 or 100 nMDNA polymerase inhibitor E; in 1×PCR buffer (50 mM Tris-HCl, pH 9, 250μM dATP, dCTP and dGTP, 500 μM dUTP, 5 mM MgCl2, 1.5 U AmpliTaq DNApolymerase, 60 nM ROX passive reference dye, 8% glycerol, 0.01%Tween-20, 0.01% NaN3, 1×SYBR Green I nucleic acid dye). A no templatecontrol was included in an eighth parallel reaction compositioncomprising the same formulation as the other seven except that there wasno cDNA and the final concentration of DNA polymerase inhibitor E was 50nM. The reaction compositions were incubated at room temperature for 15min, then thermal cycled in an ABI PRISM® 7900HT Real-Time SequenceDetection System instrument and the amplification products were analyzedon a non-denaturing agarose gel, as described in Example 2. The reactioncompositions were loaded in lanes of the gel as follows: lane B, 0 nMinhibitor E; lane C, 5 nM inhibitor E; lane d, 10 nM inhibitor E; laneE, 25 nM inhibitor E; lane F, 50 nM inhibitor E; lane G, 75 nM inhibitorE; lane H, 100 nM inhibitor E; and lane I, 50 nM inhibitor E, notemplate control.

As seen from the gel, shown in FIG. 8, the amount of desired amplicon(21) increased as the concentration of DNA polymerase inhibitorincreased until a concentration of about 75 nM. Little to no desiredamplicon was seen in the reaction composition comprising no DNApolymerase inhibitor E (lane A). The intensity of the secondary ampliconbands decreased as the DNA polymerase inhibitor concentration increased.

Example 4: Inhibiting Secondary Amplification Products Comprising PrimerDimers

Five commercially available primer pairs and corresponding TaqManreporter probes for validated gene expression assays, including assaysfor interleukin 1, beta (IL1β; assay ID Hs00174097_m1), TRAF familymember-associated NFKB activator (TANK; assay ID Hs00370305_m1), fattyacid synthase (FASN; assay ID Hs00188012_m1), solute carrier family 2,member 1 (SLC2A1; assay ID Hs00197884_m1), and phospholipase D1,phosphatidylcholine-specific (PLD1; assay ID Hs00160118_m1) wereobtained (Applied Biosystems).

To evaluate the effect of an exemplary enzyme inhibitor on the formationof primer dimer amplicons, five pairs of corresponding reactioncompositions lacking target nucleic acid were prepared in parallel. Each20 μL reaction composition pair comprised the appropriate primer pairand the corresponding TaqMan® probe at a 1× concentration; 250 μM dATP,dCTP and dGTP; 500 μM dUTP; 5 mM MgCl2; 2 U AmpliTaq DNA polymerase; 60nM ROX passive reference; 8% glycerol; 0.01% Tween-20; 0.01% NaN₃;1×SYBR Green® I in 50 mM pH 9 Tris-HCl buffer; and either 50 nMpolymerase inhibitor E or no inhibitor. The five sets of parallelreaction compositions were incubated at room temperature for 30 min andthen transferred to an ABI PRISM® 7900HT Real-Time Sequence DetectionSystem instrument. The reaction compositions were heated to 95° C. for 2min, then subjected to 40 cycles of amplification comprising 96° C. for5 sec and 60° C. for 2 min. Fifteen μL of the thermocycled reactioncompositions was loaded in individual lanes of a 4% agarose E-gel(Invitrogen) as follows: IL1β assay, lanes B (no inhibitor) and C (50 nMpolymerase inhibitor E); TANK assay, lanes D (no inhibitor) and E (50 nMpolymerase inhibitor E); FASN assay, lanes F (no inhibitor) and G (50 nMpolymerase inhibitor E); SLC2A1 assay, lanes (no inhibitor) H and I (50nM polymerase inhibitor E); and PLD1 assay, lanes J (no inhibitor) and K(50 nM polymerase inhibitor E). A molecular weight standard comprisingmarkers for 1200, 800, 400, 200, and 100 base pairs was added to lanes Aand L. The gel was electrophoresed for 15 min, and visualized bystaining with the nucleic acid dye ethidium bromide (shown in FIG. 9).The amount of undesired primer dimer product was at least reduced inreaction compositions comprising the inhibitor when compared with thecorresponding reaction composition lacking the inhibitor, e.g., comparelanes B (IL1β assay, no inhibitor) and C (IL1β assay, 50 nM polymeraseinhibitor E) or D (TANK assay, no inhibitor) and E (TANL assay, 50 nMpolymerase inhibitor E).

Example 5: Decreasing Non-Specific Fluorescence Associated with EnzymeInhibitors

To evaluate the effect of an exemplary quencher moiety of anillustrative polymerase inhibitor using PCR amplification and meltingcurve analysis, two reaction compositions were prepared. Each 20 μLreaction composition comprised primers and reporter probes from the TANKassay (described in Example 4) at a 1× concentration; 10 ng universalreference human cDNA (Stratagene); 250 μM dATP, dCTP and dGTP; 500 μMdUTP; 5 mM MgCl2; 2 U AmpliTaq DNA polymerase, 60 nM ROX passivereference, 8% glycerol, 0.01% Tween-20, 0.01% NaN3, 1×SYBR Green I in 50mM pH 9 Tris-HCl buffer and either 50 nM “polymerase inhibitor A” or 50nM polymerase inhibitor E. The reaction compositions were incubated atroom temperature for 15 min, then transferred to an ABI PRISM® 7900HTReal-Time Sequence Detection System instrument and thermocycled asdescribed in Example 4. The instrument's associated software, set atdefault conditions, was used to generate the dissociation curves for thetwo thermocycled reaction compositions, shown in FIG. 10. Twodissociation peaks were observed when the thermocycled reactioncomposition comprised “polymerase inhibitor A”, including peak A(“polymerase inhibitor A”) and peak B (the TANK amplicon). Thedissociation curve obtained with the thermocycled reaction compositioncomprising polymerase inhibitor B, by contrast, contained a peak for theTANK amplicon (shown as C in the lower panel), but no dissociation curvefor polymerase inhibitor E was readily discernible.

The enzyme inhibitors, enzyme-enzyme inhibitor complexes, methods, andkits of the current teachings have been described broadly andgenerically herein. Each of the narrower species and sub-genericgroupings falling within the generic disclosure also form part of thecurrent teachings. This includes the generic description of the currentteachings with a proviso or negative limitation removing any subjectmatter from the genus, regardless of whether or not the excised materialis specifically recited herein.

The foregoing examples are for illustration purposes and are notintended to limit the scope of the teachings herein.

Although the disclosed teachings has been described with reference tovarious enzyme inhibitors, enzyme-enzyme inhibitor complexes, methods,and kits, it will be appreciated that various changes and modificationsmay be made without departing from the teachings herein. The foregoingexamples are provided to better illustrate the present teachings and arenot intended to limit the scope of the teachings herein. Certain aspectsof the present teachings may be further understood in light of thefollowing claims.

We claim:
 1. An unbound DNA polymerase inhibitor comprising: anucleotide sequence; at least one synthetic nucleotide analog; and aquencher different from and in addition to the at least one syntheticnucleotide analog, wherein the quencher is not a nucleotide within thenucleotide sequence; wherein the nucleotide sequence comprises a firstregion, a second region, a third region, and optionally, a fourthregion; wherein the first region is complementary to the third region;wherein the first region comprises the at least one synthetic nucleotideanalog, the third region comprises the at least one synthetic nucleotideanalog, or the first region and the third region comprise the at leastone synthetic nucleotide analog; and wherein the first region comprisesa first quencher and/or the second region comprises a second quencher.2. The DNA polymerase inhibitor of claim 1, wherein the nucleotidesequence is not extendible by a DNA polymerase.
 3. The DNA polymeraseinhibitor of claim 1, further comprising a minor groove binder.
 4. TheDNA polymerase inhibitor of claim 1, wherein the at least one syntheticnucleotide analog is selected from a deaza-dA, a deaza-dG, a ddN, and acombination thereof.
 5. A kit comprising a DNA polymerase inhibitor,said inhibitor comprising a nucleotide sequence, at least one quencher,and at least one synthetic nucleotide analog different from and inaddition to the at least one quencher, wherein the at least one quencheris not a nucleotide within the nucleotide sequence.
 6. The kit of claim5, wherein the nucleotide sequence of the DNA polymerase inhibitorcomprises an aptamer.
 7. The kit of claim 5, wherein the nucleotidesequence comprises a first region, a second region, a third region, andoptionally, a fourth region; wherein the first region is complementaryto the third region.
 8. The kit of claim 7, wherein the first regioncomprises a first quencher, the second region comprises a secondquencher, or the first region comprises a first quencher and the secondregion comprises a second quencher.
 9. The kit of claim 5, wherein thenucleotide sequence comprises a first oligonucleotide and a secondoligonucleotide, wherein the first oligonucleotide comprises a firstregion and the second oligonucleotide comprises a third region andoptionally, a fourth region, and wherein the first region of the firstoligonucleotide is complementary to the third region of the secondoligonucleotide.
 10. The kit of claim 5, wherein the DNA polymeraseinhibitor further comprises a minor groove binder.
 11. The kit of claim5, further comprising a nucleic acid dye and/or a reporter probe. 12.The DNA polymerase inhibitor of claim 1, wherein the first and/or secondquencher comprises at least one of DABCYL, DABSYL, TAMRA, TET, and ROX.13. The kit of claim 7, wherein the first region, the third region, orthe first region and the third region comprise the at least onesynthetic nucleotide analog.
 14. The kit of claim 5, wherein the atleast one synthetic nucleotide analog is selected from a deaza-dA, adeaza-dG, a ddN, and a combinations thereof.
 15. The kit of claim 5,wherein the at least one quencher is a dark quencher.
 16. An unbound DNApolymerase inhibitor comprising: a nucleotide sequence; at least onesynthetic nucleotide analog comprising one or more of deaza-dA,deaza-dG, or ddN; and at least one quencher comprising one or more ofDABCYL, DABSYL, TAMRA, TET, or ROX, wherein the nucleotide sequencecomprises a first region, a second region, a third region, andoptionally, a fourth region, wherein the first region is complementaryto the third region, wherein the first region comprises the at least onesynthetic nucleotide analog, the third region comprises the at least onesynthetic nucleotide analog, or the first region and the third regioncomprise the at least one synthetic nucleotide analog, and wherein thefirst region comprises a first quencher and/or the second regioncomprises a second quencher.